Nucleic acid molecules encoding human transporter proteins

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of transporter proteinsthat are related to the chloride intracellular channel subfamily,recombinant DNA molecules, and protein production. The present inventionspecifically provides a novel human isoform of an ion channel proteinand nucleic acid molecules encoding the novel isoform, all of which areuseful in the development of human therapeutics and diagnosticcompositions and methods.

BACKGROUND OF THE INVENTION

[0002] Transporters

[0003] Transporter proteins regulate many different functions of a cell,including cell proliferation, differentiation, and signaling processes,by regulating the flow of molecules such as ions and macromolecules,into and out of cells. Transporters are found in the plasma membranes ofvirtually every cell in eukaryotic organisms. Transporters mediate avariety of cellular functions including regulation of membranepotentials and absorption and secretion of molecules and ion across cellmembranes. When present in intracellular membranes of the Golgiapparatus and endocytic vesicles, transporters, such as chloridechannels, also regulate organelle pH. For a review, see Greger, R.(1988) Annu. Rev. Physiol. 50:111-122.

[0004] Transporters are generally classified by structure and the typeof mode of action. In addition, transporters are sometimes classified bythe molecule type that is transported, for example, sugar transporters,chlorine channels, potassium channels, etc. There may be many classes ofchannels for transporting a single type of molecule (a detailed reviewof channel types can be found at Alexander, S. P. H. and J. A. Peters:Receptor and transporter nomenclature supplement. Trends Pharmacol.Sci., Elsevier, pp. 65-68 (1997) andhttp://www-biology.ucsd.edu/˜msaier/transport/titlepage2.html.

[0005] The following general classification scheme is known in the artand is followed in the present discoveries.

[0006] Channel-type transporters. Transmembrane channel proteins of thisclass are ubiquitously found in the membranes of all types of organismsfrom bacteria to higher eukaryotes. Transport systems of this typecatalyze facilitated diffusion (by an energy-independent process) bypassage through a transmembrane aqueous pore or channel without evidencefor a carrier-mediated mechanism. These channel proteins usually consistlargely of a-helical spanners, although b-strands may also be presentand may even comprise the channel. However, outer membrane porin-typechannel proteins are excluded from this class and are instead includedin class 9.

[0007] Carrier-type transporters. Transport systems are included in thisclass if they utilize a carrier-mediated process to catalyze uniport (asingle species is transported by facilitated diffusion), antiport (twoor more species are transported in opposite directions in a tightlycoupled process, not coupled to a direct form of energy other thanchemiosmotic energy) and/or symport (two or more species are transportedtogether in the same direction in a tightly coupled process, not coupledto a direct form of energy other than chemiosmotic energy).

[0008] Pyrophosphate bond hydrolysis-driven active transporters.Transport systems are included in this class if they hydrolyzepyrophosphate or the terminal pyrophosphate bond in ATP or anothernucleoside triphosphate to drive the active uptake and/or extrusion of asolute or solutes. The transport protein may or may not be transientlyphosphorylated, but the substrate is not phosphorylated.

[0009] PEP-dependent, phosphoryl transfer-driven group translocators.Transport systems of the bacterial phosphoenolpyruvate:sugarphosphotransferase system are included in this class. The product of thereaction, derived from extracellular sugar, is a cytoplasmicsugar-phosphate.

[0010] Decarboxylation-driven active transporters. Transport systemsthat drive solute (e.g., ion) uptake or extrusion by decarboxylation ofa cytoplasmic substrate are included in this class.

[0011] Oxidoreduction-driven active transporters. Transport systems thatdrive transport of a solute (e.g., an ion) energized by the flow ofelectrons from a reduced substrate to an oxidized substrate are includedin this class.

[0012] Light-driven active transporters. Transport systems that utilizelight energy to drive transport of a solute (e.g., an ion) are includedin this class.

[0013] Mechanically-driven active transporters. Transport systems areincluded in this class if they drive movement of a cell or organelle byallowing the flow of ions (or other solutes) through the membrane downtheir electrochemical gradients.

[0014] Outer-membrane porins (of b-structure). These proteins formtransmembrane pores or channels that usually allow the energyindependent passage of solutes across a membrane. The transmembraneportions of these proteins consist exclusively of b-strands that form ab-barrel. These porin-type proteins are found in the outer membranes ofGram-negative bacteria, mitochondria and eukaryotic plastids.

[0015] Methyltransferase-driven active transporters. A singlecharacterized protein currently falls into this category, theNa+-transporting methyltetrahydromethanopterin:coenzyme Mmethyltransferase.

[0016] Non-ribosome-synthesized channel-forming peptides or peptide-likemolecules. These molecules, usually chains of L- and D-amino acids aswell as other small molecular building blocks such as lactate, formoligomeric transmembrane ion channels. Voltage may induce channelformation by promoting assembly of the transmembrane channel. Thesepeptides are often made by bacteria and fungi as agents of biologicalwarfare.

[0017] Non-Proteinaceous Transport Complexes. Ion conducting substancesin biological membranes that do not consist of or are not derived fromproteins or peptides fall into this category.

[0018] Functionally characterized transporters for which sequence dataare lacking. Transporters of particular physiological significance willbe included in this category even though a family assignment cannot bemade.

[0019] Putative transporters in which no family member is an establishedtransporter. Putative transport protein families are grouped under thisnumber and will either be classified elsewhere when the transportfunction of a member becomes established, or will be eliminated from theTC classification system if the proposed transport function isdisproven. These families include a member or members for which atransport function has been suggested, but evidence for such a functionis not yet compelling.

[0020] Auxiliary transport proteins. Proteins that in some wayfacilitate transport across one or more biological membranes but do notthemselves participate directly in transport are included in this class.These proteins always function in conjunction with one or more transportproteins. They may provide a function connected with energy coupling totransport, play a structural role in complex formation or serve aregulatory function.

[0021] Transporters of unknown classification. Transport proteinfamilies of unknown classification are grouped under this number andwill be classified elsewhere when the transport process and energycoupling mechanism are characterized. These families include at leastone member for which a transport function has been established, buteither the mode of transport or the energy coupling mechanism is notknown.

[0022] Ion Channels

[0023] An important type of transporter is the ion channel. Ion channelsregulate many different cell proliferation, differentiation, andsignaling processes by regulating the flow of ions into and out ofcells. Ion channels are found in the plasma membranes of virtually everycell in eukaryotic organisms. Ion channels mediate a variety of cellularfunctions including regulation of membrane potentials and absorption andsecretion of ion across epithelial membranes. When present inintracellular membranes of the Golgi apparatus and endocytic vesicles,ion channels, such as chloride channels, also regulate organelle pH. Fora review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0024] Ion channels are generally classified by structure and the typeof mode of action. For example, extracellular ligand gated channels(ELGs) are comprised of five polypeptide subunits, with each subunithaving 4 membrane spanning domains, and are activated by the binding ofan extracellular ligand to the channel. In addition, channels aresometimes classified by the ion type that is transported, for example,chlorine channels, potassium channels, etc. There may be many classes ofchannels for transporting a single type of ion (a detailed review ofchannel types can be found at Alexander, S. P. H. and J. A. Peters(1997). Receptor and ion channel nomenclature supplement. TrendsPharmacol. Sci., Elsevier, pp. 65-68 andhttp://www-biology.ucsd.edu/˜msaier/transport/toc.html.

[0025] There are many types of ion channels based on structure. Forexample, many ion channels fall within one of the following groups:extracellular ligand-gated channels (ELG), intracellular ligand-gatedchannels (ILG), inward rectifying channels (INR), intercellular (gapjunction) channels, and voltage gated channels (VIC). There areadditionally recognized other channel families based on ion-typetransported, cellular location and drug sensitivity. Detailedinformation on each of these, their activity, ligand type, ion type,disease association, drugability, and other information pertinent to thepresent invention, is well known in the art.

[0026] Extracellular ligand-gated channels, ELGs, are generallycomprised of five polypeptide subunits, Unwin, N. (1993), Cell 72:31-41; Unwin, N. (1995), Nature 373: 37-43; Hucho, F., et al., (1996) J.Neurochem. 66: 1781-1792; Hucho, F., et al., (1996) Eur. J. Biochem.239: 539-557; Alexander, S. P. H. and J. A. Peters (1997), TrendsPharmacol. Sci., Elsevier, pp. 4-6; 36-40; 42-44; and Xue, H. (1998) J.Mol. Evol. 47: 323-333. Each subunit has 4 membrane spanning regions:this serves as a means of identifying other members of the ELG family ofproteins. ELG bind a ligand and in response modulate the flow of ions.Examples of ELG include most members of the neurotransmitter-receptorfamily of proteins, e.g., GABAI receptors. Other members of this familyof ion channels include glycine receptors, ryandyne receptors, andligand gated calcium channels.

[0027] The Voltage-Gated Ion Channel (VIC) Superfamily

[0028] Proteins of the VIC family are ion-selective channel proteinsfound in a wide range of bacteria, archaea and eukaryotes Hille, B.(1992), Chapter 9: Structure of channel proteins; Chapter 20: Evolutionand diversity. In: Ionic Channels of Excitable Membranes, 2nd Ed.,Sinaur Assoc. Inc., Pubs., Sunderland, Mass.; Sigworth, F.J. (1993),Quart. Rev. Biophys. 27: 1-40; Salkoff, L. and T. Jegla (1995), Neuron15: 489-492; Alexander, S. P. H. et al., (1997), Trends Pharmacol. Sci.,Elsevier, pp. 76-84; Jan, L. Y. et al., (1997), Annu. Rev. Neurosci. 20:91-123; Doyle, D. A, et al., (1998) Science 280: 69-77; Terlau, H. andW. Stühmer (1998), Naturwissenschaften 85: 437-444. They are often homo-or heterooligomeric structures with several dissimilar subunits (e.g.,a1-a2-d-b Ca²⁺ channels, ab₁b₂ Na⁺ channels or (a)₄-b K⁺ channels), butthe channel and the primary receptor is usually associated with the a(or a1) subunit. Functionally characterized members are specific for K⁺,Na⁺ or Ca²⁺. The K⁺ channels usually consist of homotetramericstructures with each a-subunit possessing six transmembrane spanners(TMSs). The al and a subunits of the Ca²⁺ and Na⁺ channels,respectively, are about four times as large and possess 4 units, eachwith 6 TMSs separated by a hydrophilic loop, for a total of 24 TMSs.These large channel proteins form heterotetra-unit structures equivalentto the homotetrameric structures of most K⁺ channels. All four units ofthe Ca²⁺ and Na⁺ channels are homologous to the single unit in thehomotetrameric K⁺ channels. Ion flux via the eukaryotic channels isgenerally controlled by the transmembrane electrical potential (hencethe designation, voltage-sensitive) although some are controlled byligand or receptor binding.

[0029] Several putative K⁺-selective channel proteins of the VIC familyhave been identified in prokaryotes. The structure of one of them, theKcsA K⁺ channel of Streptomyces lividans, has been solved to 3.2 Åresolution. The protein possesses four identical subunits, each with twotransmembrane helices, arranged in the shape of an inverted teepee orcone. The cone cradles the “selectivity filter” P domain in its outerend. The narrow selectivity filter is only 12 Å long, whereas theremainder of the channel is wider and lined with hydrophobic residues. Alarge water-filled cavity and helix dipoles stabilize K⁺ in the pore.The selectivity filter has two bound K⁺ ions about 7.5 Å apart from eachother. Ion conduction is proposed to result from a balance ofelectrostatic attractive and repulsive forces.

[0030] In eukaryotes, each VIC family channel type has several subtypesbased on pharmacological and electrophysiological data. Thus, there arefive types of Ca²⁺ channels (L, N, P, Q and T). There are at least tentypes of K⁺ channels, each responding in different ways to differentstimuli: voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca²⁺-sensitive[BK_(Ca), IK_(Ca) and SK_(Ca)] and receptor-coupled [K_(M) and K_(ACh)].There are at least six types of Na⁺ channels (I, II, III, μ1, H1 andPN3). Tetrameric channels from both prokaryotic and eukaryotic organismsare known in which each a-subunit possesses 2 TMSs rather than 6, andthese two TMSs are homologous to TMSs 5 and 6 of the six TMS unit foundin the voltage-sensitive channel proteins. KcsA of S. lividans is anexample of such a 2 TMS channel protein. These channels may include theK_(Na) (Na⁺-activated) and K_(VOl) (cell volume-sensitive) K⁺ channels,as well as distantly related channels such as the Tok1 K⁺ channel ofyeast, the TWIK-1 inward rectifier K⁺ channel of the mouse and theTREK-1 K⁺ channel of the mouse. Because of insufficient sequencesimilarity with proteins of the VIC family, inward rectifier K⁺ IRKchannels (ATP-regulated; G-protein-activated) which possess a P domainand two flanking TMSs are placed in a distinct family. However,substantial sequence similarity in the P region suggests that they arehomologous. The b, g and d subunits of VIC family members, when present,frequently play regulatory roles in channel activation/deactivation.

[0031] The Epithelial Na⁺ Channel (ENaC) Family

[0032] The ENaC family consists of over twenty-four sequenced proteins(Canessa, C. M., et al., (1994), Nature 367: 463-467, Le, T. and M. H.Saier, Jr. (1996), Mol. Membr. Biol. 13: 149-157; Garty, H. and L. G.Palmer (1997), Physiol. Rev. 77: 359-396; Waldmann, R., et al., (1997),Nature 386:173-177; Darboux, I., et al., (1998), J. Biol. Chem. 273:9424-9429; Firsov, D., et al., (1998), EMBO J. 17: 344-352; Horisberger,J.-D. (1998). Curr. Opin. Struc. Biol. 10: 443-449). All are fromanimals with no recognizable homologues in other eukaryotes or bacteria.The vertebrate ENaC proteins from epithelial cells cluster tightlytogether on the phylogenetic tree: voltage-insensitive ENaC homologuesare also found in the brain. Eleven sequenced C. elegans proteins,including the degenerins, are distantly related to the vertebrateproteins as well as to each other. At least some of these proteins formpart of a mechano-transducing complex for touch sensitivity. Thehomologous Helix aspersa (FMRF-amide)-activated Na⁺ channel is the firstpeptide neurotransmitter-gated ionotropic receptor to be sequenced.

[0033] Protein members of this family all exhibit the same apparenttopology, each with N- and C-termini on the inside of the cell, twoamphipathic transmembrane spanning segments, and a large extracellularloop. The extracellular domains contain numerous highly conservedcysteine residues. They are proposed to serve a receptor function.

[0034] Mammalian ENaC is important for the maintenance of Na⁺ balanceand the regulation of blood pressure. Three homologous ENaC subunits,alpha, beta, and gamma, have been shown to assemble to form the highlyNa⁺-selective channel. The stoichiometry of the three subunits isalpha₂, beta1, gamma1 in a heterotetrameric architecture.

[0035] The Glutamate-Gated Ion Channel (GIC) Family of NeurotransmitterReceptors

[0036] Members of the GIC family are heteropentameric complexes in whicheach of the 5 subunits is of 800-1000 amino acyl residues in length(Nakanishi, N., et al, (1990), Neuron 5: 569-581; Unwin, N. (1993), Cell72: 31-41; Alexander, S. P. H. and J. A. Peters (1997) Trends Pharmacol.Sci., Elsevier, pp. 36-40). These subunits may span the membrane threeor five times as putative a-helices with the N-termini (theglutamate-binding domains) localized extracellularly and the C-terminilocalized cytoplasmically. They may be distantly related to theligand-gated ion channels, and if so, they may possess substantialb-structure in their transmembrane regions. However, homology betweenthese two families cannot be established on the basis of sequencecomparisons alone. The subunits fall into six subfamilies: a, b, g, d, eand z.

[0037] The GIC channels are divided into three types: (1)a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2) kainate-and (3) N-methyl-D-aspartate (NMDA)-selective glutamate receptors.Subunits of the AMPA and kainate classes exhibit 35-40% identity witheach other while subunits of the NMDA receptors exhibit 22-24% identitywith the former subunits. They possess large N-terminal, extracellularglutamate-binding domains that are homologous to the periplasmicglutamine and glutamate receptors of ABC-type uptake permeases ofGram-negative bacteria. All known members of the GIC family are fromanimals. The different channel (receptor) types exhibit distinct ionselectivities and conductance properties. The NMDA-selective largeconductance channels are highly permeable to monovalent cations andCa²⁺. The AMPA- and kainate-selective ion channels are permeableprimarily to monovalent cations with only low permeability to Ca²⁺.

[0038] The Chloride Channel (ClC) Family

[0039] The ClC family is a large family consisting of dozens ofsequenced proteins derived from Gram-negative and Gram-positivebacteria, cyanobacteria, archaea, yeast, plants and animals (Steinmeyer,K., et al., (1991), Nature 354: 301-304; Uchida, S., et al., (1993), J.Biol. Chem. 268: 3821-3824; Huang, M.-E., et al., (1994), J. Mol. Biol.242: 595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604; Fisher,W. E., et al., (1995), Genomics. 29:598-606; and Foskett, J. K. (1998),Annu. Rev. Physiol. 60: 689-717). These proteins are essentiallyubiquitous, although they are not encoded within genomes of Haemophilusinfluenzae, Mycoplasma genitalium, and Mycoplasma pneumoniae. Sequencedproteins vary in size from 395 amino acyl residues (M. jannaschii) to988 residues (man). Several organisms contain multiple ClC familyparalogues. For example, Synechocystis has two paralogues, one of 451residues in length and the other of 899 residues. Arabidopsis thalianahas at least four sequenced paralogues, (775-792 residues), humans alsohave at least five paralogues (820-988 residues), and C. elegans alsohas at least five (810-950 residues). There are nine known members inmammals, and mutations in three of the corresponding genes cause humandiseases. E. coli, Methanococcus jannaschii and Saccharomyces cerevisiaeonly have one ClC family member each. With the exception of the largerSynechocystis paralogue, all bacterial proteins are small (395-492residues) while all eukaryotic proteins are larger (687-988 residues).These proteins exhibit 10-12 putative transmembrane a-helical spanners(TMSs) and appear to be present in the membrane as homodimers. While onemember of the family, Torpedo ClC-O, has been reported to have twochannels, one per subunit, others are believed to have just one.

[0040] All functionally characterized members of the ClC familytransport chloride, some in a voltage-regulated process. These channelsserve a variety of physiological functions (cell volume regulation;membrane potential stabilization; signal transduction; transepithelialtransport, etc.). Different homologues in humans exhibit differing anionselectivities, i.e., ClC4 and ClC5 share a NO₃ ⁻>Cl^(−>Br) ⁻>I⁻conductance sequence, while ClC3 has an I⁻>Cl⁻ selectivity. The ClC4 andClC5 channels and others exhibit outward rectifying currents withcurrents only at voltages more positive than +20 mV.

[0041] Chloride Intracellular Channel (CLIC)

[0042] The novel human protein, and encoding gene, provided by thepresent invention is a novel isoform of chloride intracellular channel 5(CLIC5) (Genbank gi8393147). Specifically, the isoform of the presentinvention differs from the art-known CLIC5 isoform at the 3′ end. Theisoform provided by the present invention, having a novel 3′ end, issupported by EST data (see FIG. 2). Furthermore, the 3′ end of the cDNAmolecule of the present invention is intact after the stop codon, andthe stop codon and polyA signal are present in the genomic sequence.

[0043] CLIC5 has been isolated from placental microvilli, where itexists as a component of a multimeric complex consisting of actin,ezrin, alpha-actinin, gelsolin, IQGAP1, and other known cytoskeletalproteins. CLIC5 is enriched in placental microvilli compared with theCLIC1 isoform and CLIC5 is associated with the detergent-insolublecytoskeletal fraction of microvilli. CLIC5 is concentrated within theapical region of the trophoblast. It has been suggested that CLIC5 playsa distinct role in chloride transport compared with the CLIC1 and CLIC4isoforms, and that CLIC5 interacts with the cortical actin cytoskeletonin polarized epithelial cells (Berryman et al., Mol Biol Cell 2000May;11(5):1509-21).

[0044] Intracellular voltage gated ion channels reside in the cellularorganelles. They regulate membrane potentials of intracellular membranesand plasma membrane. These proteins may be extracted from microsomes andnuclei. Intracellular ion channels belong to the CLIC family ofchannels. These proteins are represented by several isoforms inmammalian genomes. Higher levels of CLIC expression is observed in theheart, kidney, and skeletal muscle.

[0045] CLICs play an essential role in transepithelial ion transport.Increasing cell volume (osmotic swelling) activates some chloridechannels. Intestinal chloride channels can be vital for a number ofphysiological processes, such as digestion, electrolyte homeostasis andperistalsis. Possible interaction of CLICs with other components of thetransepithelial transport machine, such as CFTR or cystic fibrosistransmembrane conductance regulator, may shed light on the mechanisms ofdiseases associated with impaired water/ion balance. The role of CLICsin acidification of intracellular vesicles may be crucial for virusassembly and propagation.

[0046] Interestingly, the intracellular ion channels, and CLICs inparticular, may be essential for cell division and apoptosis (celldeath). Although their roles in the cell cycle are not well documentedat this time, there is a possibility they can be up-regulated in rapidlydividing cells such as cancer cells. In this case, synthetic CLICmediators may be delivered to the transformed tissue in order to slowcancerous growth.

[0047] At least three inherited diseases are associated with mutationsin chloride channels: myotonia congenita, Dent's disease, and Bartter'ssyndrome. The sequences provided by the present invention can be used toscreen human populations for allele variations and associated disorders.Inhibitors or activators of the protein of the present invention can beused as therapeutic agents to treat diseases of the digestive tract.

[0048] For a further review of chloride intracellular channel, see Li etal., J Biol Chem Sep. 5, 2000; Clarke et al., Am J Physiol GastrointestLiver Physiol 2000 July;279(1):G132-8; Franco-Obregon et al., Biophys J2000 July;79(1):202-14; and Waldegger et al., J Am Soc Nephrol 2000July;11(7):1331-9.

[0049] Animal Inward Rectifier K⁺ Channel (IRK-C) Family

[0050] IRK channels possess the “minimal channel-forming structure” withonly a P domain, characteristic of the channel proteins of the VICfamily, and two flanking transmembrane spanners (Shuck, M. E., et al.,(1994), J. Biol. Chem. 269: 24261-24270; Ashen, M. D., et al., (1995),Am. J. Physiol. 268: H506-H511; Salkoff, L. and T. Jegla (1995), Neuron15: 489-492; Aguilar-Bryan, L., et al., (1998), Physiol. Rev. 78:227-245; Ruknudin, A., et al., (1998), J. Biol. Chem. 273: 14165-14171).They may exist in the membrane as homo- or heterooligomers. They have agreater tendency to let K⁺ flow into the cell than out.Voltage-dependence may be regulated by external K⁺, by internal Mg²⁺ byinternal ATP and/or by G-proteins. The P domains of IRK channels exhibitlimited sequence similarity to those of the VIC family, but thissequence similarity is insufficient to establish homology. Inwardrectifiers play a role in setting cellular membrane potentials, and theclosing of these channels upon depolarization permits the occurrence oflong duration action potentials with a plateau phase. Inward rectifierslack the intrinsic voltage sensing helices found in VIC family channels.In a few cases, those of Kir1.1a and Kir6.2, for example, directinteraction with a member of the ABC superfamily has been proposed toconfer unique functional and regulatory properties to the heteromericcomplex, including sensitivity to ATP. The SURI sulfonylurea receptor(spQ09428) is the ABC protein that regulates the Kir6.2 channel inresponse to ATP, and CFTR may regulate Kir1.1a. Mutations in SUR1 arethe cause of familial persistent hyperinsulinemic hypoglycemia ininfancy (PHHI), an autosomal recessive disorder characterized byunregulated insulin secretion in the pancreas.

[0051] ATP-Gated Cation Channel (ACC) Family

[0052] Members of the ACC family (also called P2X receptors) respond toATP, a functional neurotransmitter released by exocytosis from manytypes of neurons (North, R. A. (1996), Curr. Opin. Cell Biol. 8:474-483; Soto, F., M. Garcia-Guzman and W. Stühmer (1997), J. Membr.Biol. 160: 91-100). They have been placed into seven groups (P2X₁-P2X₇)based on their pharmacological properties. These channels, whichfunction at neuron-neuron and neuron-smooth muscle junctions, may playroles in the control of blood pressure and pain sensation. They may alsofunction in lymphocyte and platelet physiology. They are found only inanimals.

[0053] The proteins of the ACC family are quite similar in sequence(>35% identity), but they possess 380-1000 amino acyl residues persubunit with variability in length localized primarily to the C-terminaldomains. They possess two transmembrane spanners, one about 30-50residues from their N-termini, the other near residues 320-340. Theextracellular receptor domains between these two spanners (of about 270residues) are well conserved with numerous conserved glycyl and cysteylresidues. The hydrophilic C-termini vary in length from 25 to 240residues. They resemble the topologically similar epithelial Na⁺ channel(ENaC) proteins in possessing (a) N- and C-termini localizedintracellularly, (b) two putative transmembrane spanners, (c) a largeextracellular loop domain, and (d) many conserved extracellular cysteylresidues. ACC family members are, however, not demonstrably homologouswith them. ACC channels are probably hetero- or homomultimers andtransport small monovalent cations (Me⁺). Some also transport Ca²⁺; afew also transport small metabolites.

[0054] The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca2+ Channel(RIR-CaC) Family

[0055] Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate(IP3)-sensitive Ca²⁺-release channels function in the release of Ca²⁺from intracellular storage sites in animal cells and thereby regulatevarious Ca²⁺-dependent physiological processes (Hasan, G. et al., (1992)Development 116: 967-975; Michikawa, T., et al., (1994), J. Biol. Chem.269: 9184-9189; Tunwell, R. E. A., (1996), Biochem. J. 318: 477-487;Lee, A. G. (1996) Biomembranes, Vol. 6, Transmembrane Receptors andChannels (A. G. Lee, ed.), JAI Press, Denver, CO., pp 291-326;Mikoshiba, K., et al., (1996) J. Biochem. Biomem. 6: 273-289). Ryreceptors occur primarily in muscle cell sarcoplasmic reticular (SR)membranes, and IP3 receptors occur primarily in brain cell endoplasmicreticular (ER) membranes where they effect release of Ca²⁺ into thecytoplasm upon activation (opening) of the channel.

[0056] The Ry receptors are activated as a result of the activity ofdihydropyridine-sensitive Ca²⁺ channels. The latter are members of thevoltage-sensitive ion channel (VIC) family. Dihydropyridine-sensitivechannels are present in the T-tubular systems of muscle tissues.

[0057] Ry receptors are homotetrameric complexes with each subunitexhibiting a molecular size of over 500,000 daltons (about 5,000 aminoacyl residues). They possess C-terminal domains with six putativetransmembrane a-helical spanners (TMSs). Putative pore-forming sequencesoccur between the fifth and sixth TMSs as suggested for members of theVIC family. The large N-terminal hydrophilic domains and the smallC-terminal hydrophilic domains are localized to the cytoplasm. Lowresolution 3-dimensional structural data are available. Mammals possessat least three isoforms that probably arose by gene duplication anddivergence before divergence of the mammalian species. Homologues arepresent in humans and Caenorabditis elegans.

[0058] IP₃ receptors resemble Ry receptors in many respects. (1) Theyare homotetrameric complexes with each subunit exhibiting a molecularsize of over 300,000 daltons (about 2,700 amino acyl residues). (2) Theypossess C-terminal channel domains that are homologous to those of theRy receptors. (3) The channel domains possess six putative TMSs and aputative channel lining region between TMSs 5 and 6. (4) Both the largeN-terminal domains and the smaller C-terminal tails face the cytoplasm.(5) They possess covalently linked carbohydrate on extracytoplasmicloops of the channel domains. (6) They have three currently recognizedisoforms (types 1, 2, and 3) in mammals which are subject todifferential regulation and have different tissue distributions.

[0059] IP₃ receptors possess three domains: N-terminal IP₃-bindingdomains, central coupling or regulatory domains and C-terminal channeldomains. Channels are activated by IP₃ binding, and like the Ryreceptors, the activities of the IP₃ receptor channels are regulated byphosphorylation of the regulatory domains, catalyzed by various proteinkinases. They predominate in the endoplasmic reticular membranes ofvarious cell types in the brain but have also been found in the plasmamembranes of some nerve cells derived from a variety of tissues.

[0060] The channel domains of the Ry and IP₃ receptors comprise acoherent family that in spite of apparent structural similarities, donot show appreciable sequence similarity of the proteins of the VICfamily. The Ry receptors and the IP₃ receptors cluster separately on theRIR-CaC family tree. They both have homologues in Drosophila. Based onthe phylogenetic tree for the family, the family probably evolved in thefollowing sequence: (1) A gene duplication event occurred that gave riseto Ry and IP₃ receptors in invertebrates. (2) Vertebrates evolved frominvertebrates. (3) The three isoforms of each receptor arose as a resultof two distinct gene duplication events. (4) These isoforms weretransmitted to mammals before divergence of the mammalian species.

[0061] The Organellar Chloride Channel (O-CIC) Family

[0062] Proteins of the O-CIC family are voltage-sensitive chloridechannels found in intracellular membranes but not the plasma membranesof animal cells (Landry, D, et al., (1993), J. Biol. Chem. 268:14948-14955; Valenzuela, Set al., (1997), J. Biol. Chem. 272:12575-12582; and Duncan, R. R., et al., (1997), J. Biol. Chem. 272:23880-23886).

[0063] They are found in human nuclear membranes, and the bovine proteintargets to the microsomes, but not the plasma membrane, when expressedin Xenopus laevis oocytes. These proteins are thought to function in theregulation of the membrane potential and in transepithelial ionabsorption and secretion in the kidney. They possess two putativetransmembrane a-helical spanners (TMSs) with cytoplasmic N- andC-termini and a large luminal loop that may be glycosylated. The bovineprotein is 437 amino acyl residues in length and has the two putativeTMSs at positions 223-239 and 367-385. The human nuclear protein is muchsmaller (241 residues). A C. elegans homologue is 260 residues long.

[0064] Transporter proteins, particularly members of the chlorideintracellular channel subfamily, are a major target for drug action anddevelopment. Accordingly, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize previously unknown transportproteins. The present invention advances the state of the art byproviding previously unidentified human transport proteins.

SUMMARY OF THE INVENTION

[0065] The present invention is based in part on the identification ofamino acid sequences of a novel human chloride intracellular channelisoform, as well as allelic variants and other mammalian orthologsthereof. These unique peptide sequences, representing a novel isoform,and nucleic acid sequences that encode these peptides, can be used asmodels for the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate ion channelactivity in cells and tissues that express the isoform. Experimentaldata as provided in FIG. 1 indicates expression in humans in embryos,placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytomacells, and fetal brain.

DESCRIPTION OF THE FIGURE SHEETS

[0066]FIG. 1 provides the nucleotide sequence of a cDNA molecule thatencodes the transporter protein of the present invention. (SEQ ID NO: 1)In addition structure and functional information is provided, such asATG start, stop and tissue distribution, where available, that allowsone to readily determine specific uses of inventions based on thismolecular sequence. Experimental data as provided in FIG. 1 indicatesexpression in humans in embryos, placenta, uterus and ovary tumors, eye(lens), testis, pheochromocytoma cells, and fetal brain.

[0067]FIG. 2 provides the predicted amino acid sequence of thetransporter of the present invention. (SEQ ID NO:2) In additionstructure and functional information such as protein family, function,and modification sites is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence.

[0068]FIG. 3 provides genomic sequences that span the gene encoding thetransporter protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 116different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0069] General Description

[0070] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a transporter protein or part of atransporter protein and are related to the chloride intracellularchannel subfamily. Utilizing these sequences, additional genomicsequences were assembled and transcript and/or cDNA sequences wereisolated and characterized. Based on this analysis, the presentinvention provides amino acid sequences of a novel human chlorideintracellular channel isoform (interchangeably referred to herein as theisoform, transporter, or ion channel of the present invention), nucleicacid sequences in the form of transcript/cDNA sequences and genomicsequences that encode this isoform, nucleic acid variation (allelicinformation), tissue distribution of expression, and information aboutthe closest art known protein/peptide/domain that has structural orsequence homology to the isoform of the present invention.

[0071] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known transporter proteins ofthe chloride intracellular channel subfamily and the expression patternobserved. Experimental data as provided in FIG. 1 indicates expressionin humans in embryos, placenta, uterus and ovary tumors, eye (lens),testis, pheochromocytoma cells, and fetal brain. The art has clearlyestablished the commercial importance of members of this family ofproteins and proteins that have expression patterns similar to that ofthe present gene. Some of the more specific features of the peptides ofthe present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and in the annotationprovided in the Figures, and/or are known within the art for each of theknown chloride intracellular channel family or subfamily of transporterproteins.

[0072] Specific Embodiments

[0073] Peptide Molecules

[0074] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thetransporter family of proteins and are related to the chlorideintracellular channel subfamily (protein sequences are provided in FIG.2, transcript/cDNA sequences are provided in FIGS. 1 and genomicsequences are provided in FIG. 3). The peptide sequences provided inFIG. 2, as well as the obvious variants described herein, particularlyallelic variants as identified herein and using the information in FIG.3, will be referred herein as the transporter peptides of the presentinvention, transporter peptides, or peptides/proteins of the presentinvention.

[0075] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprising theamino acid sequences of the transporter peptides disclosed in the FIG.2, (encoded by the nucleic acid molecule shown in FIG. 1,transcript/cDNA or FIG. 3, genomic sequence), as well as all obviousvariants of these peptides that are within the art to make and use. Someof these variants are described in detail below.

[0076] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0077] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0078] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thetransporter peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0079] The isolated transporter peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in embryos, placenta, uterus and ovary tumors, eye (lens),testis, pheochromocytoma cells, and fetal brain. For example, a nucleicacid molecule encoding the transporter peptide is cloned into anexpression vector, the expression vector introduced into a host cell andthe protein expressed in the host cell. The protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Many of these techniques are describedin detail below.

[0080] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided inFIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein isprovided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

[0081] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0082] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genonic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the transporter peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0083] The transporter peptides of the present invention can be attachedto heterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a transporter peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the transporter peptide. “Operativelylinked” indicates that the transporter peptide and the heterologousprotein are fused in-frame. The heterologous protein can be fused to theN-terminus or C-terminus of the transporter peptide.

[0084] In some uses, the fusion protein does not affect the activity ofthe transporter peptide per se. For example, the fusion protein caninclude, but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant transporter peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

[0085] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A transporter peptide-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the transporter peptide.

[0086] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0087] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the transporter peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0088] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0089] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A.M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

[0090] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0091] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the transporter peptides of the present invention as well asbeing encoded by the same genetic locus as the transporter peptideprovided herein. The gene encoding the novel transporter protein of thepresent invention is located on a genome component that has been mappedto human chromosome 6 (as indicated in FIG. 3), which is supported bymultiple lines of evidence, such as STS and BAC map data.

[0092] Allelic variants of a transporter peptide can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of the transporterpeptide as well as being encoded by the same genetic locus as thetransporter peptide provided herein. Genetic locus can readily bedetermined based on the genomic information provided in FIG. 3, such asthe genomic sequence mapped to the reference human. The gene encodingthe novel transporter protein of the present invention is located on agenome component that has been mapped to human chromosome 6 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data. As used herein, two proteins (or a regionof the proteins) have significant homology when the amino acid sequencesare typically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a transporter peptideencoding nucleic acid molecule under stringent conditions as more fullydescribed below.

[0093]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 116 different nucleotide positions. Some of theseSNPs, which are located in introns and 3′ of the ORF, may affectcontrol/regulatory elements.

[0094] Paralogs of a transporter peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the transporter peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a transporter peptide encoding nucleic acid molecule under moderateto stringent conditions as more fully described below.

[0095] Orthologs of a transporter peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the transporter peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a transporter peptide encoding nucleicacid molecule under moderate to stringent conditions, as more fullydescribed below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

[0096] Non-naturally occurring variants of the transporter peptides ofthe present invention can readily be generated using recombinanttechniques. Such variants include, but are not limited to deletions,additions and substitutions in the amino acid sequence of thetransporter peptide. For example, one class of substitutions areconserved amino acid substitution. Such substitutions are those thatsubstitute a given amino acid in a transporter peptide by another aminoacid of like characteristics. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr; exchange of the acidic residues Asp and Glu; substitutionbetween the amide residues Asn and Gln; exchange of the basic residuesLys and Arg; and replacements among the aromatic residues Phe and Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

[0097] Variant transporter peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind ligand, abilityto transport ligand, ability to mediate signaling, etc. Fully functionalvariants typically contain only conservative variation or variation innon-critical residues or in non-critical regions. FIG. 2 provides theresult of protein analysis and can be used to identify criticaldomains/regions. Functional variants can also contain substitution ofsimilar amino acids that result in no change or an insignificant changein function. Alternatively, such substitutions may positively ornegatively affect function to some degree.

[0098] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0099] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as transporter activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J Mol Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0100] The present invention further provides fragments of thetransporter peptides, in addition to proteins and peptides that compriseand consist of such fragments, particularly those comprising theresidues identified in FIG. 2. The fragments to which the inventionpertains, however, are not to be construed as encompassing fragmentsthat may be disclosed publicly prior to the present invention.

[0101] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a transporter peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the transporter peptide or could be chosenfor the ability to perform a function, e.g. bind a substrate or act asan immunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe transporter peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0102] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally intransporter peptides are described in basic texts, detailed monographs,and the research literature, and they are well known to those of skillin the art (some of these features are identified in FIG. 2).

[0103] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racernization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0104] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins - Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N. Acad. Sci. 663:48-62(1992)).

[0105] Accordingly, the transporter peptides of the present inventionalso encompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature transporter peptide is fused withanother compound, such as a compound to increase the half-life of thetransporter peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature transporter peptide, suchas a leader or secretory sequence or a sequence for purification of themature transporter peptide or a pro-protein sequence.

[0106] Protein/Peptide Uses

[0107] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in atransporter-effector protein interaction or transporter-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

[0108] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0109] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, transporters isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the transporter. Experimental data asprovided in FIG. 1 indicates that the transporter proteins of thepresent invention are expressed in humans in embryos, placenta, uterusand ovary tumors, eye (lens), testis, and pheochromocytoma cells, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in fetal brain. A largepercentage of pharmaceutical agents are being developed that modulatethe activity of transporter proteins, particularly members of thechloride intracellular channel subfamily (see Background of theInvention). The structural and functional information provided in theBackground and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in humans in embryos, placenta, uterusand ovary tumors, eye (lens), testis, pheochromocytoma cells, and fetalbrain. Such uses can readily be determined using the informationprovided herein, that known in the art and routine experimentation.

[0110] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to transporters that arerelated to members of the chloride intracellular channel subfamily. Suchassays involve any of the known transporter functions or activities orproperties useful for diagnosis and treatment of transporter-relatedconditions that are specific for the subfamily of transporters that theone of the present invention belongs to, particularly in cells andtissues that express the transporter. Experimental data as provided inFIG. 1 indicates that the transporter proteins of the present inventionare expressed in humans in embryos, placenta, uterus and ovary tumors,eye (lens), testis, and pheochromocytoma cells, as indicated by virtualnorthern blot analysis. In addition, PCR-based tissue screening panelsindicate expression in fetal brain. The proteins of the presentinvention are also useful in drug screening assays, in cell-based orcell-free systems ((Hodgson, Bio/technology, 1992, September10(9);973-80). Cell-based systems can be native, i.e., cells thatnormally express the transporter, as a biopsy or expanded in cellculture. Experimental data as provided in FIG. 1 indicates expression inhumans in embryos, placenta, uterus and ovary tumors, eye (lens),testis, pheochromocytoma cells, and fetal brain. In an alternateembodiment, cell-based assays involve recombinant host cells expressingthe transporter protein.

[0111] The polypeptides can be used to identify compounds that modulatetransporter activity of the protein in its natural state or an alteredform that causes a specific disease or pathology associated with thetransporter. Both the transporters of the present invention andappropriate variants and fragments can be used in high-throughputscreens to assay candidate compounds for the ability to bind to thetransporter. These compounds can be further screened against afunctional transporter to determine the effect of the compound on thetransporter activity. Further, these compounds can be tested in animalor invertebrate systems to determine activity/effectiveness. Compoundscan be identified that activate (agonist) or inactivate (antagonist) thetransporter to a desired degree.

[0112] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the transporter protein and a molecule that normally interactswith the transporter protein, e.g. a substrate or a component of thesignal pathway that the transporter protein normally interacts (forexample, another transporter). Such assays typically include the stepsof combining the transporter protein with a candidate compound underconditions that allow the transporter protein, or fragment, to interactwith the target molecule, and to detect the formation of a complexbetween the protein and the target or to detect the biochemicalconsequence of the interaction with the transporter protein and thetarget, such as any of the associated effects of signal transductionsuch as changes in membrane potential, protein phosphorylation, cAMPturnover, and adenylate cyclase activation, etc.

[0113] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0114] One candidate compound is a soluble fragment of the receptor thatcompetes for ligand binding. Other candidate compounds include mutanttransporters or appropriate fragments containing mutations that affecttransporter function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, isencompassed by the invention.

[0115] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) transporter activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate transporter activity. Thus, the transport of aligand, change in cell membrane potential, activation of a protein, achange in the expression of genes that are up- or down-regulated inresponse to the transporter protein dependent signal cascade can beassayed.

[0116] Any of the biological or biochemical functions mediated by thetransporter can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the transporter can be assayed.Experimental data as provided in FIG. 1 indicates that the transporterproteins of the present invention are expressed in humans in embryos,placenta, uterus and ovary tumors, eye (lens), testis, andpheochromocytoma cells, as indicated by virtual northern blot analysis.In addition, PCR-based tissue screening panels indicate expression infetal brain.

[0117] Binding and/or activating compounds can also be screened by usingchimeric transporter proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a ligand-binding region can be usedthat interacts with a different ligand then that which is recognized bythe native transporter. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the transporter is derived.

[0118] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the transporter (e.g. binding partners and/orligands). Thus, a compound is exposed to a transporter polypeptide underconditions that allow the compound to bind or to otherwise interact withthe polypeptide. Soluble transporter polypeptide is also added to themixture. If the test compound interacts with the soluble transporterpolypeptide, it decreases the amount of complex formed or activity fromthe transporter target. This type of assay is particularly useful incases in which compounds are sought that interact with specific regionsof the transporter. Thus, the soluble polypeptide that competes with thetarget transporter region is designed to contain peptide sequencescorresponding to the region of interest.

[0119] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the transporter protein, or fragment, orits target molecule to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay.

[0120] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of transporter-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a transporter-binding protein and a candidate compound are incubatedin the transporter protein-presenting wells and the amount of complextrapped in the well can be quantitated. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the transporter protein target molecule, or which arereactive with transporter protein and compete with the target molecule,as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0121] Agents that modulate one of the transporters of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

[0122] Modulators of transporter protein activity identified accordingto these drug screening assays can be used to treat a subject with adisorder mediated by the transporter pathway, by treating cells ortissues that express the transporter. Experimental data as provided inFIG. 1 indicates expression in humans in embryos, placenta, uterus andovary tumors, eye (lens), testis, pheochromocytoma cells, and fetalbrain. These methods of treatment include the steps of administering amodulator of transporter activity in a pharmaceutical composition to asubject in need of such treatment, the modulator being identified asdescribed herein.

[0123] In yet another aspect of the invention, the transporter proteinscan be used as “bait proteins” in a two-hybrid assay or three-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the transporter and are involved in transporteractivity. Such transporter-binding proteins are also likely to beinvolved in the propagation of signals by the transporter proteins ortransporter targets as, for example, downstream elements of atransporter-mediated signaling pathway. Alternatively, suchtransporter-binding proteins are likely to be transporter inhibitors.

[0124] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a transporterprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming atransporter-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the transporter protein.

[0125] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a transporter-modulating agent, an antisensetransporter nucleic acid molecule, a transporter-specific antibody, or atransporter-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

[0126] The transporter proteins of the present invention are also usefulto provide a target for diagnosing a disease or predisposition todisease mediated by the peptide. Accordingly, the invention providesmethods for detecting the presence, or levels of, the protein (orencoding mRNA) in a cell, tissue, or organism. Experimental data asprovided in FIG. 1 indicates expression in humans in embryos, placenta,uterus and ovary tumors, eye (lens), testis, pheochromocytoma cells, andfetal brain. The method involves contacting a biological sample with acompound capable of interacting with the transporter protein such thatthe interaction can be detected. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

[0127] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0128] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered transporter activity incell-based or cell-free assay, alteration in ligand or antibody-bindingpattern, altered isoelectric point, direct amino acid sequencing, andany other of the known assay techniques useful for detecting mutationsin a protein. Such an assay can be provided in a single detection formator a multi-detection format such as an antibody chip array.

[0129] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0130] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the transporter protein in which oneor more of the transporter functions in one population is different fromthose in another population. The peptides thus allow a target toascertain a genetic predisposition that can affect treatment modality.Thus, in a ligand-based treatment, polymorphism may give rise to aminoterminal extracellular domains and/or other ligand-binding regions thatare more or less active in ligand binding, and transporter activation.Accordingly, ligand dosage would necessarily be modified to maximize thetherapeutic effect within a given population containing a polymorphism.As an alternative to genotyping, specific polymorphic peptides could beidentified.

[0131] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in embryos, placenta, uterus and ovary tumors, eye(lens), testis, pheochromocytoma cells, and fetal brain. Accordingly,methods for treatment include the use of the transporter protein orfragments.

[0132] Antibodies

[0133] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0134] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0135] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0136] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0137] Antibodies are preferably prepared from regions or discretefragments of the transporter proteins. Antibodies can be prepared fromany region of the peptide as described herein. However, preferredregions will include those involved in function/activity and/ortransporter/binding partner interaction. FIG. 2 can be used to identifyparticularly important regions while sequence alignment can be used toidentify conserved and unique sequence fragments.

[0138] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0139] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, 131 ^(I),³⁵S or ³H.

[0140] Antibody Uses

[0141] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the transporter proteins of the present invention are expressed inhumans in embryos, placenta, uterus and ovary tumors, eye (lens),testis, and pheochromocytoma cells, as indicated by virtual northernblot analysis. In addition, PCR-based tissue screening panels indicateexpression in fetal brain. Further, such antibodies can be used todetect protein in situ, in vitro, or in a cell lysate or supernatant inorder to evaluate the abundance and pattern of expression. Also, suchantibodies can be used to assess abnormal tissue distribution orabnormal expression during development or progression of a biologicalcondition. Antibody detection of circulating fragments of the fulllength protein can be used to identify turnover.

[0142] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in embryos, placenta, uterus and ovarytumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. Ifa disorder is characterized by a specific mutation in the protein,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant protein.

[0143] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin embryos, placenta, uterus and ovary tumors, eye (lens), testis,pheochromocytoma cells, and fetal brain. The diagnostic uses can beapplied, not only in genetic testing, but also in monitoring a treatmentmodality. Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

[0144] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0145] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in embryos,placenta, uterus and ovary tumors, eye (lens), testis, pheochromocytomacells, and fetal brain. Thus, where a specific protein has beencorrelated with expression in a specific tissue, antibodies that arespecific for this protein can be used to identify a tissue type.

[0146] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the transporter peptide to abinding partner such as a ligand or protein binding partner. These usescan also be applied in a therapeutic context in which treatment involvesinhibiting the protein's function. An antibody can be used, for example,to block binding, thus modulating (agonizing or antagonizing) thepeptides activity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. See FIG. 2 for structuralinformation relating to the proteins of the present invention.

[0147] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nucleic acid arrays and similar methods have been developedfor antibody arrays.

[0148] Nucleic Acid Molecules

[0149] The present invention further provides isolated nucleic acidmolecules that encode a transporter peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the transporter peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0150] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. However, there can besome flanking nucleotide sequences, for example up to about 5KB, 4KB,3KB, 2KB, or 1KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0151] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0152] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0153] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

[0154] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0155] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ IDNO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can compriseseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0156] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0157] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0158] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the transporter peptidealone, the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0159] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0160] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the transporter proteinsof the present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0161] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0162] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0163] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0164] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel transporter protein of the present invention is located on agenome component that has been mapped to human chromosome 6 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data.

[0165]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 116 different nucleotide positions. Some of theseSNPs, which are located in introns and 3′ of the ORF, may affectcontrol/regulatory elements.

[0166] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2× SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0167] Nucleic Acid Molecule Uses

[0168] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, SNPs were identified at 116 differentnucleotide positions.

[0169] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0170] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0171] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0172] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0173] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. The gene encoding the noveltransporter protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 6 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data.

[0174] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0175] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the MRNA produced from thenucleic acid molecules described herein.

[0176] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0177] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0178] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0179] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the transporter proteins of the present invention areexpressed in humans in embryos, placenta, uterus and ovary tumors, eye(lens), testis, and pheochromocytoma cells, as indicated by virtualnorthern blot analysis. In addition, PCR-based tissue screening panelsindicate expression in fetal brain.

[0180] Accordingly, the probes can be used to detect the presence of, orto determine levels of, a specific nucleic acid molecule in cells,tissues, and in organisms. The nucleic acid whose level is determinedcan be DNA or RNA. Accordingly, probes corresponding to the peptidesdescribed herein can be used to assess expression and/or gene copynumber in a given cell, tissue, or organism. These uses are relevant fordiagnosis of disorders involving an increase or decrease in transporterprotein expression relative to normal results.

[0181] In vitro techniques for detection of MRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

[0182] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a transporter protein, such asby measuring a level of a transporter-encoding nucleic acid in a sampleof cells from a subject e.g., mRNA or genomic DNA, or determining if atransporter gene has been mutated. Experimental data as provided in FIG.1 indicates that the transporter proteins of the present invention areexpressed in humans in embryos, placenta, uterus and ovary tumors, eye(lens), testis, and pheochromocytoma cells, as indicated by virtualnorthern blot analysis. In addition, PCR-based tissue screening panelsindicate expression in fetal brain.

[0183] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate transporter nucleic acid expression.

[0184] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the transporter gene, particularly biological andpathological processes that are mediated by the transporter in cells andtissues that express it. Experimental data as provided in FIG. 1indicates expression in humans in embryos, placenta, uterus and ovarytumors, eye (lens), testis, pheochromocytoma cells, and fetal brain. Themethod typically includes assaying the ability of the compound tomodulate the expression of the transporter nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired transporter nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the transporter nucleic acidor recombinant cells genetically engineered to express specific nucleicacid sequences.

[0185] The assay for transporter nucleic acid expression can involvedirect assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the signal pathway. Further, theexpression of genes that are up- or down-regulated in response to thetransporter protein signal pathway can also be assayed. In thisembodiment the regulatory regions of these genes can be operably linkedto a reporter gene such as luciferase.

[0186] Thus, modulators of transporter gene expression can be identifiedin a method wherein a cell is contacted with a candidate compound andthe expression of mRNA determined. The level of expression oftransporter mRNA in the presence of the candidate compound is comparedto the level of expression of transporter mRNA in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of nucleic acid expression based on this comparison and beused, for example to treat a disorder characterized by aberrant nucleicacid expression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

[0187] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate transporter nucleic acidexpression in cells and tissues that express the transporter.Experimental data as provided in FIG. 1 indicates that the transporterproteins of the present invention are expressed in humans in embryos,placenta, uterus and ovary tumors, eye (lens), testis, andpheochromocytoma cells, as indicated by virtual northern blot analysis.In addition, PCR-based tissue screening panels indicate expression infetal brain. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) ornucleic acid expression.

[0188] Alternatively, a modulator for transporter nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the transporter nucleic acid expression in the cells andtissues that express the protein. Experimental data as provided in FIG.1 indicates expression in humans in embryos, placenta, uterus and ovarytumors, eye (lens), testis, pheochromocytoma cells, and fetal brain.

[0189] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe transporter gene in clinical trials or in a treatment regimen. Thus,the gene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0190] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in transporter nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in transporter genes andgene expression products such as mRNA. The nucleic acid molecules can beused as hybridization probes to detect naturally occurring geneticmutations in the transporter gene and thereby to determine whether asubject with the mutation is at risk for a disorder caused by themutation. Mutations include deletion, addition, or substitution of oneor more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the transporter geneassociated with a dysfinction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a transporterprotein.

[0191] Individuals carrying mutations in the transporter gene can bedetected at the nucleic acid level by a variety of techniques. FIG. 3provides information on SNPs that have been found in the gene encodingthe transporter protein of the present invention. SNPs were identifiedat 116 different nucleotide positions. Some of these SNPs, which arelocated in introns and 3′ of the ORF, may affect control/regulatoryelements. The gene encoding the novel transporter protein of the presentinvention is located on a genome component that has been mapped to humanchromosome 6 (as indicated in FIG. 3), which is supported by multiplelines of evidence, such as STS and BAC map data. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way. In some uses, detection of themutation involves the use of a probe/primer in a polymerase chainreaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), suchas anchor PCR or RACE PCR, or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080(1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter ofwhich can be particularly useful for detecting point mutations in thegene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0192] Alternatively, mutations in a transporter gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0193] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0194] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant transporter gene and a wild-type gene can be determined by directDNA sequencing. A variety of automated sequencing procedures can beutilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101 ; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159(1993)).

[0195] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0196] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the transporter gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at116 different nucleotide positions. Some of these SNPs, which arelocated in introns and 3′ of the ORF, may affect control/regulatoryelements.

[0197] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0198] The nucleic acid molecules are thus useful as antisenseconstructs to control transporter gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of transporter protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of MRNA into transporter protein.

[0199] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of transporter nucleicacid. Accordingly, these molecules can treat a disorder characterized byabnormal or undesired transporter nucleic acid expression. Thistechnique involves cleavage by means of ribozymes containing nucleotidesequences complementary to one or more regions in the MRNA thatattenuate the ability of the mRNA to be translated. Possible regionsinclude coding regions and particularly coding regions corresponding tothe catalytic and other functional activities of the transporterprotein, such as ligand binding.

[0200] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in transporter geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredtransporter protein to treat the individual.

[0201] The invention also encompasses kits for detecting the presence ofa transporter nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the transporter proteins of thepresent invention are expressed in humans in embryos, placenta, uterusand ovary tumors, eye (lens), testis, and pheochromocytoma cells, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in fetal brain. For example,the kit can comprise reagents such as a labeled or labelable nucleicacid or agent capable of detecting transporter nucleic acid in abiological sample; means for determining the amount of transporternucleic acid in the sample; and means for comparing the amount oftransporter nucleic acid in the sample with a standard. The compound oragent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to detect transporter proteinmRNA or DNA.

[0202] Nucleic Acid Arrays

[0203] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

[0204] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0205] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0206] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0207] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0208] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The ARNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0209] Using such arrays, the present invention provides methods toidentify the expression of the transporter proteins/peptides of thepresent invention. In detail, such methods comprise incubating a testsample with one or more nucleic acid molecules and assaying for bindingof the nucleic acid molecule with components within the test sample.Such assays will typically involve arrays comprising many genes, atleast one of which is a gene of the present invention and or alleles ofthe transporter gene of the present invention. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at116 different nucleotide positions. Some of these SNPs, which arelocated in introns and 3′ of the ORF, may affect control/regulatoryelements.

[0210] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol.3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0211] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0212] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0213] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0214] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified transporter gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0215] Vectors/Host Cells

[0216] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0217] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0218] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in procaryotic or eukaryoticcells or in both (shuttle vectors).

[0219] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0220] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0221] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0222] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0223] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0224] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0225] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0226] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0227] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterotransporter. Typical fusionexpression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein. Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology185:60-89 (1990)).

[0228] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0229] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0230] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0231] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J 6:187-195 (1987)).

[0232] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0233] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0234] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0235] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0236] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0237] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0238] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0239] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell- free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0240] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such astransporters, appropriate secretion signals are incorporated into thevector. The signal sequence can be endogenous to the peptides orheterologous to these peptides.

[0241] Where the peptide is not secreted into the medium, which istypically the case with transporters, the protein can be isolated fromthe host cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0242] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0243] Uses of Vectors and Host Cells

[0244] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga transporter protein or peptide that can be further purified to producedesired amounts of transporter protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0245] Host cells are also useful for conducting cell-based assaysinvolving the transporter protein or transporter protein fragments, suchas those described above as well as other formats known in the art.Thus, a recombinant host cell expressing a native transporter protein isuseful for assaying compounds that stimulate or inhibit transporterprotein function.

[0246] Host cells are also useful for identifying transporter proteinmutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutanttransporter protein (for example, stimulating or inhibiting function)which may not be indicated by their effect on the native transporterprotein.

[0247] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA that is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a transporterprotein and identifying and evaluating modulators of transporter proteinactivity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

[0248] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the transporter proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0249] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the transporter protein to particularcells.

[0250] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0251] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0252] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0253] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect ligand binding,transporter protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivotransporter protein function, including ligand interaction, the effectof specific mutant transporter proteins on transporter protein functionand ligand interaction, and the effect of chimeric transporter proteins.It is also possible to assess the effect of null mutations, that ismutations that substantially or completely eliminate one or moretransporter protein functions.

[0254] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 5 1 2033 DNA Human 1 cgcggatcct gtgacacctc cgggcagccc ggcacttgttgctcccacga cctgttgtca 60 ttcccttaac ccggctttcc ccgtggcccc ccgcctcctcccggcttcgc tccttttcat 120 gtgagcatct gggacactga tctctcagac cccgctgctcgggctggaga atagatggtt 180 ttgtgaaaaa ttaaacaccg ccctgaagag gagccccgctgggcagcggc aggagcgcag 240 agtgctggcc caggtgctgc agaggtggcg cctccccggcccgggacggt agccccgggc 300 gccaacggca tgacagactc ggcgacagct aacggggacgacagggaccc cgagatcgag 360 ctctttgtga aggctggaat cgatggagaa agcatcggcaactgtccttt ctctcagcgc 420 ctcttcatga tcctctggct gaaaggagtc gtgttcaatgtcaccactgt ggatctgaaa 480 agaaagccag ctgacctgca caacctagcc cccggcacgcacccgccctt cctgaccttc 540 aacggggacg tgaagacaga cgtcaataag atcgaggagttcctggagga gaccttgacc 600 cctgaaaagt accccaaact ggctgcaaaa caccgggaatccaacacagc gggcatcgac 660 atcttttcca agttttctgc ctacatcaaa aataccaagcagcagaacaa tgctgctctt 720 gaaagaggcc taaccaaggc tctaaagaaa ttggatgactacctgaacac ccctctacca 780 gaggagattg acgccaacac ttgtggggaa gacaaggggtcccggcgcaa gttcctggat 840 ggggatgagc tgaccctggc tgactgcaat ctgttgcccaagctccatgt ggtcaaggta 900 agagagctct acccacaggg gcctgcaaga tccagctccatcttaggccc aggtcacctg 960 tgtggatgag tcaaggacag taccacctgt tggtcaagaacctggaccct gaagtcaggt 1020 aataaggacc caaggtcacc ctctgctgct tgttggctgtgtggcctcct tgagcttcag 1080 ttttcattta taaaataggc atatattgct tacttcaagtgttggtggaa gagtaaatac 1140 agcgtgaaag tgcttggcat attgtggggg cttaatatgtgtaatagtcg caattatcgt 1200 tgttgtatac agtcatatca ctccaaaggc ctcttcctcataggatttcc ctggctacac 1260 ccctacagct ctattaaatg tgccctcata tgcattttttctttgtgcac agacccacct 1320 tctcacttcc tccagcaact tcctaaggtg agcccacattattttcctca tctatcaaat 1380 gaagaggtgg aggttgcaag aagtgatgtc actttcttgctatcattgca cttactaacc 1440 atttgcagca tgtagtgtca tcctctccta tataacaaaccctgggaatc tgagagttgg 1500 aaaggacatt tagaggtcat ccaaaacaat ctcccacttcaacctggacc actttctgct 1560 gtttctgtga caggcttctg tgaagctgtg actgcagcctctgagaacga ggagccctct 1620 gcttaatgag ccagccctac catgttagat agctctgattattaaatcat tgttctttac 1680 aatgagccca agcatgcctc cctgctatcc attttttctctagagtaaca gagaacagct 1740 ttgcttgcct tcacctcatt tgaagacagt agttgtatccccctaagctc tgtcaatgag 1800 cacttcttcc cccattcttt cctgaccctc gtcagcctagtatcagatag ccatactgtg 1860 ctctatttta cgcatgctta tatcttactg tctcagccagacagcaaact ctgtgaggaa 1920 aggacctttt taaagtgtga tggtgggcac acagtggccattcaataaat actcattgat 1980 tgatcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaa 2033 2 219 PRT Human 2 Met Thr Asp Ser Ala Thr Ala AsnGly Asp Asp Arg Asp Pro Glu Ile 1 5 10 15 Glu Leu Phe Val Lys Ala GlyIle Asp Gly Glu Ser Ile Gly Asn Cys 20 25 30 Pro Phe Ser Gln Arg Leu PheMet Ile Leu Trp Leu Lys Gly Val Val 35 40 45 Phe Asn Val Thr Thr Val AspLeu Lys Arg Lys Pro Ala Asp Leu His 50 55 60 Asn Leu Ala Pro Gly Thr HisPro Pro Phe Leu Thr Phe Asn Gly Asp 65 70 75 80 Val Lys Thr Asp Val AsnLys Ile Glu Glu Phe Leu Glu Glu Thr Leu 85 90 95 Thr Pro Glu Lys Tyr ProLys Leu Ala Ala Lys His Arg Glu Ser Asn 100 105 110 Thr Ala Gly Ile AspIle Phe Ser Lys Phe Ser Ala Tyr Ile Lys Asn 115 120 125 Thr Lys Gln GlnAsn Asn Ala Ala Leu Glu Arg Gly Leu Thr Lys Ala 130 135 140 Leu Lys LysLeu Asp Asp Tyr Leu Asn Thr Pro Leu Pro Glu Glu Ile 145 150 155 160 AspAla Asn Thr Cys Gly Glu Asp Lys Gly Ser Arg Arg Lys Phe Leu 165 170 175Asp Gly Asp Glu Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu 180 185190 His Val Val Lys Val Arg Glu Leu Tyr Pro Gln Gly Pro Ala Arg Ser 195200 205 Ser Ser Ile Leu Gly Pro Gly His Leu Cys Gly 210 215 3 106323 DNAHuman misc_feature (1)...(106323) n = A,T,C or G 3 gtagtgagga catctttcccttttctttgc ctatcagatg cctgaccttt caggaaggtt 60 cagctaaagt gccttttgctccatgaagcc ttttcctctc ctcaccccaa ccatggtaaa 120 tagtatcttt acgtgacagcttgtatagct ccttcatctt cccaattcat gtggcagcct 180 tagttttcat ttcatgtaactattattctg aaagggattt tgatctctgt taagaccgtt 240 aaaaccatgc ctttgtttcctcaaagtgtg cacttccata gtgtgcacta agaaaagcag 300 tttgtagagc agttgagaggacaggctcaa gtctgtctgg gtttaaatcc tgacttcacc 360 atttcctagc tgaatgactagggataagtt acttaaatac ttcatgtctc agtttcccca 420 cttgtaaaat tgggatcattatactatttg taaatattat acaagttcac acatgtaaag 480 tgctcaacat ttttgcctagcacataataa gtattattac ttattttttt attaagcgat 540 aaatttttta ataaagctattaaacaaata catgttttta gtaaggaagg acatagcaca 600 aacaggatta attaaagagaaggcaaagaa gacaaagaga attccaccag attctggtgc 660 aaaacattca ataattattttttaaagaac tgaagggtgg gaatgtctgt tggtgcaacc 720 actttagaaa acctatggcagtgtttacac aaggagagca ttcttataca ctacaactca 780 gcaagtccac tcctaagtatacatccaaga gaaaagtgtc tgtgaatcca accaaaagat 840 gtttgataga gagaatgttcgtgacagagt agtcatgata gtcacctgca caaatgtcca 900 ttgacagaag aatgaataagtaagtgtgtc ggtgaaaatg gatgatacac aactacacat 960 agcacaatgg atgaatctcacaaacatagt aatgagcgaa agaagccaga caccaacatt 1020 ccaggcaaaa agatcctatgctgctacaag ccagaataat ggttaccctg ggacagtgga 1080 gacgggagaa aagatgtggagaggagagag caggaggagc ttctagggct gtggctaaca 1140 ttttattctt catctggcgctcgttacatg ggtgggttgg attgtgaaaa ttcattgagg 1200 catacaccta agatatgtgcatcttttctg tgtttgcatc ctacttcact aaaacaacca 1260 gaagagaacg ggaatgaagtaagatcagaa aataaaggaa gaaatagaaa gaaaatgaaa 1320 gaagggagaa gaaaaataaacaaacccaga aggctgaaag gcatggaagc cgcacctgac 1380 cgggttgact cacccctctgccccaggagc cacccctgcc ctgccctgcc cgtccccagc 1440 accctactct ccttttgcttagcccaagga tggcaaatgc cccttttggt gggggttctg 1500 agcatcacta agacaggaagggctttccaa gtgggtaagg ctggtagtgg agtaactcgg 1560 aggagctgac gctttattaaagaaacagaa gacttgagag ccatctcctc tcttccttcc 1620 ccttcccctt tccccttcctcttccccttc ccctttcccc ttcctcttcc ccttcccctt 1680 tccccttcct cttccccttcccctttcccc ttcctcttcc ccttcccctt tccccttcct 1740 cttccccttc cccttctcctcgactctttc tgccttcgcc tcattatccg cccccaggac 1800 atcctcttgg ccttggtggctttgccagga ccagctctgt ctccgcctcc tgccctgcgg 1860 ggtacttggg gcggggggccgtggcccggc ccaggcccga gctgccgggg tgtccccgcc 1920 gcgtccccgc ccggctgaccccgccctgca gccgccggct attttgggcg cgttggcggc 1980 ggcgggatcg ctgacagtcgcggatcctgt gacacctccg ggcagcccgg cacttgttgc 2040 tcccacgacc tgttgtcattcccttaaccc ggctttcccc gtggcccccc gcctcctacc 2100 cggcttcgct ccttttcatgtgagcatgct gggacactga tctctcagac cccgctgctc 2160 gggctggaga atagatggttttgtgaaaaa ttaaacaccg ccctgaagag gagccccgct 2220 gggcagcggc aggtagcgcagagtgctggc ccaggtgctg cagaggtggc gcctccccgg 2280 cccgggacgg tagccccgggcgccaacggc atgacagact cggcgacagc taacggggac 2340 gacagggacc ccgagatcgagctctttgtg aaggtaggtc ggggtccagt ctcccgccgc 2400 acctgccgcg cctcccgccgggctcaccca ccagggctcg gggtgcgctg gactccggac 2460 cctcccagga gggcggcgcggagccctgtg ctcccccgtg gcctttccgc aggccagcgc 2520 cccgctgcga ggctcctgggcggaatggag gaaggcaggt tccggcaggt ggccgtcttc 2580 tcggagagtt gatccgcagtttgtccaccc ccgtccccgc ctccgctgtc accgctgcga 2640 gtccgagtcc agagcccagcccggacgcga ttcaggctgg tcctgaaaga cgtctccagg 2700 tgtcgggatg cggcagaaacctggctggac ccgtgaacct ttcctttaaa acgggaggag 2760 ggggaggaat gggagagaaacgttattgac acaatgatct ttggcaaagc gaaaatgaca 2820 tttcttaaaa ttctctttctctctttcgct cagagatcct aaaataaaag ccaaggagac 2880 aaaagtgttt tcttgcttggtgacttgcaa agcagaaaac aagcactcca ttctccgacg 2940 ttattatttt tgtcataatcatgatttttc aaggcagtat caaggaaatg ttaggcgttt 3000 ggtagtaggg agagcctcccgttcaacctg atgcagacag ggatgaagga gcacagtgca 3060 aattaaatga agtcaccttgctccttggac agaaactgcc taatgtcaga gctgctgtac 3120 tttatttgca acctgtaggaggaaggatgg aggcgaggct gcggcggcgt ttcccggtcc 3180 gccggtgtaa ggagaaagaaacatttgatc agcccttggt gccgacgctg cagaagtagg 3240 gaaacctgag gctgcgggagagtagcctcc agggctgagc agaaacagtc gccaggactt 3300 gggggagggg aagtcggggactgcctgggc gccaagacct ttcacaaagg gggaaaactc 3360 tctcagtgca aagtttccctagaaatatgc agatcgctga gaagggaaga gtcactggtg 3420 cctgtcaggg gcctcctggcagaatttaaa aggcataaat attccctgga attgggaaaa 3480 tgttattttg cccaaaagatccttctctac aagctaatta aatagatgtt tgcaaaaagt 3540 atgagatcta cataaaccttgataacaatc tgtggtcttt ggtactttct gactcttaaa 3600 atgtgactta ctttacacttggtacgtaat ctgtctccgt tgtcccagcc taccacaaag 3660 gtttgttaaa tttcttctgctgggttttac attctttgtg tttatttttc aagcttacac 3720 ctgacattta gtgacacttgctttcttcag cctgttctct tcccactgtg gcccctgtgt 3780 gctcctttgg tttctgggactccccccacc cccaccccag gctggatttc caggtccttt 3840 ggcagaccca ggtaataaaggaggtcattt ctcgtctgat gctgggtagt gttggacctt 3900 agggcagcaa gggggtgttctgaaccccag catccaggcc tgggccctag gggtgaatag 3960 ggagagcttt aacaaagcgatgagtcttgc tgctctcctt gaagaaagcg attctaggac 4020 ttactcagat tccttgagagtggtagtttc aaggtgtcaa gtttctgaaa gatatttgaa 4080 acacaaatag gttctttagaaccaaaggag ccctttgccc cactcctagg tgtggagacc 4140 gattctacct gcacctttggaattttcttg ttggcagtgt tggagagtct ctccatttat 4200 ataaaaacct tactgcacaatttgcagcgt tccccaaata attctaaaat gactgtgttg 4260 tcatttgttt tttccctaaaggaaatcaat gagagccagg caggccttca tggctagctc 4320 aaaaaccaga ttgatggggagatcaaaaca tcaggtatga aagttgtttt gactcctccc 4380 tcaaccccat ctttttgagcttcaggttag cagttctcat ttacaagtaa atgggacaag 4440 taaaggctag aaatattgtgaaataaagat gctgcccagg ccttacttga actgtgtcat 4500 ttgagggaag caatatccttgttgacagaa tcacactttt ccaaagtttg gagtttctct 4560 gaggtacatg attccccataaaaagaaagg ccttcttgtt gatggtgtca ggtaactgaa 4620 gctctttctg cagggcagggtgggttggtt tctatgaaga cttccaagaa ggatgggtcc 4680 cacccagtta tagtcatgacttggtcattt tcaacacccc cttggaagga cctcccaacc 4740 tcttgtttcc actgggtgtaccactctaaa tcacagtcac ctaatgagtt tcacaatgac 4800 atgtaattgt aattgtctcccacgtcaatt gatagtcatt atctgttact actactgctg 4860 ataatgataa tgataagaagaaggaaagat tgcagcatat taggacataa taagtatgtt 4920 caaatacctt atctcatttgatcatcagca actttataag gcttgattca cttatgtgaa 4980 agataaagaa aacaaataaactggattcac aggtacctta tgagactcgc tgtcagagca 5040 cttgatgagc tgcagtttgctcttcttgta acagcccagg gaactaagaa tgataattcc 5100 tcctgttctg ttgaacaatacatgtgctca cagaaagtag gtaaaatgcc caaaggtaga 5160 tggcagatag cattcagatccagtcctcaa atctagcagt ttagactttg aatttcttgc 5220 tcttttccaa atacctatatgtcaggcctg ataccttatt ttgccaagtt aggctttcaa 5280 gtgccatcat ggtgacatttatttatgtta ttaatccatt ctaatgtgtt catagaatgc 5340 ttccaaggag actaacccatttctgctatg tactccgagt ctttcccttt cggggaacat 5400 tctgacagtg acttttaagagattatttcc ccacattttt agtttctaca ataagaaatg 5460 ttagatcatg tcaactctgattctaatgtc ccataatacc tgtggatggc gcagggattt 5520 ggagatgcag tacctgagttcaagtcttgc ctctgcctct aattagctaa atgatcctga 5580 acaaattatt taactttcaggagcctcact ggccctctta aaacaagagc atggttataa 5640 ataaggagac catataatttattgtccaaa tctagacact tttgagagtg aaagggaatg 5700 atatcaataa ttaggctggaaccacaggta taacccagga tagtttggga gcaaataagg 5760 atgtgtggtc attctggttatgaaaatact gtcattgtag atttgctgtg aggattccat 5820 gggagaacat aggtgaaaacactttgtaaa ttgaaaattg ctgtaaacac actgacttga 5880 atattatgag tcccatggcagactgaacct ttctggacct gcacgtccct gatttgcctt 5940 ggggaacaaa ggcctggctccttcctcact tgcagggata ttttgagagt aaatgagata 6000 atatgtagct tggactttgatcctaagagg aggacaacag gctagaggta atggcagtga 6060 gtgtggttta ttgaaagaaatagagcaagg aagtaaaagc aaaacagaaa gcaatttgaa 6120 aagacaaaca caggatgtgctgtgtaggtt agatcaaggc attccaccca tttgcattgc 6180 ttcctccact cccagtctcacccctggacc ccagaagtga ctaatgatac agacaagctc 6240 gccgcaagtg tctcttcagccaggaactga ggtttcaaca gaaaccagtg gttctcagcc 6300 ttcactacct attcaatccattgagagaga ttttaaaact tctgatggca gtttgctccc 6360 taagccgatt acatcagtgtgtctgtgggt gggaccctga aacaatattt tcatatccac 6420 tctcaggtgg ctccagcgtgcagccctggt ggagagccac tgagtcataa tcagaggcag 6480 gtccaatagc aggcagcttctgccaccgct gacataggtg gccttgctaa tgctgattaa 6540 atgtccagaa cttctaacagcctcagggca tcacggtcag aagtgttgtt gatagaaagc 6600 ttgcatccaa tcactgcccagttaacagat gctgacgaac tattaagccc tatccacagg 6660 actccagaac aatcgtgtatagggttggtc tagtcaaaga atcatcaatt cagcatgggg 6720 gaagtaaaac agagttctgctaattgtctg atggaaacga ctagtctttc atttattgag 6780 tggcccaaaa gtttctgagtggtgcagggt gcaatacaag ttgtggtgac tcgtggtgtg 6840 gtcatctgac ttccagaacagataggatca gatcttgtgg tgtcgcctct atgtccctga 6900 gctactgtga ctgcctggtcaccttgggcc gcttgcctaa gcatgagctc actaggtttt 6960 taaagttccc ttcatcatattaagctactg tgtttttctt tcccactatc ttcaggtttt 7020 gatctgtagt caataacgcaatacacaaat ttgcctgccc tgctatggca gtgagtgctt 7080 cttctcctgg taacacatccctggagagct agcgtcaaga gttgtatgat gcatatttga 7140 agtgccatgg cagggtggtgggaatgagga ctgggtctgc agaagctgac gtccactgcc 7200 agtgttgaga gctcctgcatccctgatgac ttgtgttgtc tctttgggca tttattggcc 7260 ccctccatgc aagacaaagcgtctgtcaat ttagacagtc agttaggtca aaaacttctc 7320 attcttggtt ccctttgagttgtcagctgc tggccccatg ttagtggtgg caagagatgt 7380 ggtggaagag tgaaactttggtgtcaggct cgggttcacc agtgtgttgg ccaggatggg 7440 ctgcttgacc tctctggaccacagattttt tttttgtttt aaccagtgca gtgctaatag 7500 cagcagcaca tctttcatggagttggtgtc agcatttcaa gctgagtatg gatctggcct 7560 attagtaagt ctttctctgtaggctccatg tagcccaata agtaaaagta aagcccttgg 7620 gcttccctcc cttgatgtgtacaagaagtc agaggctcag tcctaccagc tgaatctcac 7680 atctcagaac caagatacttcttgaaagaa aatgtaagtt tccaaagcag gtcgtcattc 7740 agggaactgg aagagaaatgttcccgattg ttggagctga cccctgagaa cacaagcagt 7800 gagtctcatt gctgcagaccaatggcaact gaagtacagt gcaaccacca ctggatctga 7860 ctgcctctga ctggactgctgctccataag ctggagccca ctagagccca tgtctggagt 7920 aatgtggttg gactgactacagggtaactg cagatccctg acccactggg ttcttcaaac 7980 tcaggggaag ttgattcctcctgtcagttg ctccctcatc caagaaagct cctacataac 8040 tgcttcccca aggctcctggtagccaggcc agggtaaagg atcctcttta ttttcaagag 8100 gatccctggt tctctttgcagattttacta ccccttcctc aacaatcttc cagctatgca 8160 tagccatagg tgctctcatcaaatatactc acacatgcac acgtattatc taccaggatt 8220 atggggaaaa tgtcgtaggctctcactctc tggtatctat taaaaaacta ttacccagaa 8280 cacatcctga gttgtatctgttgtcaagac tggaggaact agagatagta aagatgcaaa 8340 gtggttaagc atgagtttcctaagatgtag ttctgtaaag gactctctct acccagggtg 8400 tcttaaatct aacactgcttgtgttctgag gggtcagctc ccacagtcag ggacatttct 8460 cttccagttc gctaaatgacaacctgcttt ggaaacgtgt cttttctttc aagaaagaag 8520 tatctaagtt ctgagatgtgaaattcttta aggacaaata agataaatag cctactgtct 8580 gaactgaaac gaattcttccctgtaaaaag aaatggagtt gttaacttct agaagttctt 8640 ttagctctgc tggcatgggaagagtcttta agggggattc aaagactctt cccatgccag 8700 cagcactgga ggtaaaccatgggtaggctg gtggttctca gcctttgggg catgaaagag 8760 tcactgcact tgccttcttcacttgtacct gcccaggtcc tgtctataga gtttcagatt 8820 taggagggtg gggtgaggcccaggcatctg tatgcttaag aagctaccca ctggtgattc 8880 tgaagcccat caaagactgagaaccactgg tgcaaaccat gggtaaatag ggctgaacta 8940 ctcagtggag catcaaaaggacaaaatatg gtccagtcgc tacttcaaac aagcatttca 9000 caggatggag gtagccaaggcaatatgctg gttcacatga atcactccag attgacctca 9060 aataaaccag aaaagggaggaaagaaatgc ttttgacctt tcttttgtaa tttttctttt 9120 cttcctttgt atattttttattatatagga gtgacacata attttaaatg gcaatgcatg 9180 aaggagatag taagataaagaatgtaggga gttggggata aaatataatc aatcaatcaa 9240 tatatgaaaa tgcttacagagcacctactc tgtgccaggc tctgtgaaag ctgaaggggg 9300 ctgggggaga tcagagaagaagacaggctc cagtagcttg tgagagtgcc actgaggagt 9360 gcaatgcaaa caagcatcagtggcacatga gtgacaggaa tgcaatgagt catgcatatg 9420 atgggaccag tgtggcccaaatggtgtctt caggcccagc tttgggtagg gtgggacctg 9480 agttgagtct tgaagaatgatcaggattca gaggtggcca aaagtgagca accatgtcag 9540 gtccagcaaa ggtgtggagatggggatgct cagggcttat gagtacacgg ggttgtctga 9600 agcaagtggt atatgaaaggagcaattgga aataggacag caaaaaagcc aacaacagtg 9660 gcaggagggg gccttgaatgggaggtaagt ggtatctgct cccttgtaag tagtggggag 9720 taactggaag tgtctacacacaataggaga ttgacttggg ttatgtttag aaagattgat 9780 agctggactt cgtgctttagtgggaaaatc aatgaactca aagtatgatg gataccctaa 9840 aagtcctgac ttgatcactgcacaatctat gcatgtaaca aaattgctca tgtaccccat 9900 aaatttgtgc aaatgaaaatcgggaaaaaa aaaaaaagaa aaagaaaaaa aacaggaaaa 9960 aacaaacaaa caaaagaaagaagatctgga ttccaggtct gccacgcact ggttgcgtga 10020 cttgctttaa gggtaagttttttttttcta taaaatgtga tcaacattca taaaaccata 10080 gaatttatta tttggaaaagcttccagaag tcatccagtc tgaccttctc tgatgagata 10140 agttcccaat ctaacactttgggatatacg ctatgtgggt ttaaggaaga gtagtgagat 10200 aaaatgtgag ctgctgctgtttttgccatt gctattacgg ctgcattcac tgtgaccatg 10260 ttggaataag gagagccctgaggcagaagt cagtgaggag gctatcccag tggtcccatc 10320 aggaggccaa gtgagtccaaacaagggcca aaagacagag tgtgttgtgg aatacagaag 10380 tgaatacaaa attaggtgcatgccttggaa gaagcctgtg caaatagtgt atattagggg 10440 caccaacacc gaagactcagcagtttcctg gtacattcac ccctggaaag gttagtgtcc 10500 acatctagaa tggtgatatctttgatgggc ttgaccatta ctgtctaaga tattgtaaat 10560 tgaagagtac agacaaactaattgagcttt aagtttcctt ttaactctga agaaagccta 10620 tgtatcctgt gaggaagggagttgctgttc attaaaatcg tttgatttgg aatggtgtaa 10680 tctgaagtgc tggcaggccagtcctgtgca gatgttcctt agaatgctga aggatcagag 10740 ctgaggatca caggggctcagggctaggga ttttatctga acttctgctc agagctacag 10800 gtgacggagg aggttccccagaggagggag catggagaag aagtacagaa ataaggactt 10860 gctttaaaaa atgtcttttggggctgggca tggtggctca tgactgtaat ctcagcactt 10920 tgggaggccg aggcagggggatcatgtcag gggatcgaga ccatcctggc caacatggtg 10980 aaacgccgtc tctagcaaaatacaaaaaaa ttagccgggt gtggtggcac ccacctatag 11040 ccccagctac ttaggagactgaggcagggg aatcgcttga acccgggagg tggatgttgc 11100 agagggctga gtttgcaccactgtactcca gcctggtgac agagcaagac tgtctcaaaa 11160 aaaaaaaaaa ggaaaaaaaagaaatatcat ttgagaggga ggaatgaggc acgtgaggag 11220 ttagccaagg tgatcaaggaataagaggta ggaaataaat gcaatgtcct aggcaaagga 11280 aatcatgtga acaatggctcagaagcaaga caagcccgga tgtgtcccag aaaactcaga 11340 gctctgcttg gctgtgctgaagagggtgtg aatggatatt gggaaggcag ataaaaatgt 11400 aagttagggg ccaggcgcagtggctcacat cagtaatccc agcactttgg gaggctgagg 11460 tgggcagatc atgaggtcaagagttcgaga ccagcctggc caatatggtg aaaccccgtc 11520 tctactaaag atacaaaaaattagctggac atggtggcgc atgcctgtaa tcccagctac 11580 tcaggaagtt gaggcaggagaattgcttga accaaggagg cggaggctgc agtgggccaa 11640 gattgcgcca ttgcactccaggctgggcga tagggcaaga ttccatctaa atatatatat 11700 atatatatat atatatatatatatgtattt atacatgtgt atatatatat atatatatgt 11760 atttatacat gtgtatatatatatttatac atgtatatat atatttatac atgtatatat 11820 atttatacat gtgtgtatatatatatatat aaatatatat atatatatat acacacatag 11880 gctctgtcct aatctctttgagctgctatg acaaaatgcc atgaactagg tagcttataa 11940 acaacagaaa tttatttctcacagttctgg aggctgggga gtctatcagt gcaccaatag 12000 atttggtgtc tggtgagggccacaatgtgg ttcatagctg tcaccttctc actgggttct 12060 cacattgtgg aggaggagaactctattctc ttcagcccct catgagaaca ctaatcccat 12120 ttgtgagggc tccaccctcatgacctaatc acctcccaaa gcctccacct gctaatgcca 12180 acacctcaga gatgaaattttagcatagga attctggggg gacacaaaca ttcagatcta 12240 gcagatcaga tcatgaagcatctctgatgg caagctgggg gatccagact tttgtttata 12300 aagtggaaga ctgctgtgcccctacctgcc tgtggagcaa ttgtagttta aaagagggaa 12360 gtgcttaatt atctaaacatggtgcatttg gccctgcatc tgaggcaggg tgttttggcg 12420 tgggaggcct ttgcagcacgtctctggtta cccacaggct ttggtgtgta gaagttttgt 12480 gccagttacc ccatatcctatatcaacctc tatggacttc agggtgatgg tttcttttcc 12540 acttttgttt aacgttattctatggtaaga gggaaaagaa acaagtgtcc gactggagaa 12600 ataacacaaa aggggaaggaggggaggatg tgcagtgaat ggagtccaac tgggaaccca 12660 ttacctagaa tagagacgaagtaacaccat gtcccagtta ttctcttaaa catccaattt 12720 gggagattgt catggcaacagatagaggtt tcttgatcca gcccaaggct cctctcctgt 12780 caatgtccac gtctgcttcctaatgctgcc agtgtgcatg ggccccgtca acaccaagga 12840 tgaagacgag gggccgtctcacctgccttt tattcattta cacatttgct ctctgtggca 12900 ttttatagtt atagcataaaatctagagac agcagtgtct ttagaatttg cccctgaaac 12960 caaaggagcc atatgtccttggccctttgt cacaaatgac tcaggtttta cttgtggaat 13020 tgaaggttct gggcagggcttccccacagg ctctgggaga ggggagctgt gtagggaaag 13080 ttatgtggcc tttcctcattcaggtacttt gtgaatataa cagggatctg atctgtgtgg 13140 caggattatt attatgaagtcaaataagat aagaaacatg aacccctttt gcaaactaca 13200 gtcttcactg ttcagatgtcaggcactgcc ttggtcggta taggtcaact gctgaaacaa 13260 caacctacca actttttcacacagtagctc aatgcagtgt ttagtgagtg gtctccccca 13320 cttggactcc ctccaccctgtagttctgcc accctgcggg acggccaact cctttgctgg 13380 gctctctaca gtcagccagcacagggaaga ttgaagttgg aggacatcat ggtaggtttg 13440 catggtcgaa tcacttccacccactttcca tagtccagag ctcaatcctg tggctgttcc 13500 tctgtgcaag ggaagctgggaagtgtcatc taactgtgct gcaaggacag agaggaagca 13560 ggtttggagc tagtctctaccgtagaaact aagttatgta ttcttgtttc ctactacaaa 13620 ctggcatccc acctgcagcctctctcacct cctgttgacc atgcttaata ctgtccactt 13680 aatcttccta aagaagaaccttctcctgta tgacatcgat tccccatctg ctagatcaac 13740 ttcaactgct tccacctgatactgaaacta ttcttgcatt ccttttccca gcgttatttc 13800 ccatattctg ttaaaatggttccttgtgtg tacatacagt tttttgaagc atttttgggt 13860 ctcttatcct ttgaggaaggaagatagtta atcctctcat ataccaggtg atttgtcagg 13920 ggctgcatga tgggttgctctatacaaact tagaaacctg gagatccgga ctccagacct 13980 gaccactctc caccagaacagggtttccca gccttgcaca ggcatgagaa ccacctgggg 14040 cattttccaa actccagcccaaatgtgtga atcagagtct tcagggaagg aggtcccaga 14100 tacctgtaat ttaacaagtgctgcaggtga ttcttgtgcc cagtctggtt aagggaacac 14160 tgcccttaac ctaaccaggccccatgggag tatccaaaac ccacaggggt gagtctaaca 14220 gtggaaattt aaagccctgtgacattcaaa tgtaggagag atttggtagg cgagtggtct 14280 tctactgctc tcaggtcaccccaaggtata taacatctca gtcattttat atagtgacct 14340 ttagacttag gatctgctccatggcccgtg gaattataaa aacagttgca gccttgttta 14400 ttgttttgtt gttggtgttgttgttgtttt tgcaaaggta tttatcccca tcctcaaagt 14460 ataaggttct tgaagaggaaaatgttggga aagagatagc ggggatggaa gagggaaaag 14520 ggtaggtgag acagaggatcactgaagtgg cttatgatac agaaaaacca gtcttcacac 14580 ctcagtcctc tttggtcccatgagtggtgt ctttgcattt tcaaaatcag agaatcagtt 14640 tggaaattga ataatagcaacagtggctaa ctgctattct gaacacctta gtttcgctga 14700 ttccttgaat tccatggagtaggtcttggt actattccca atttacagat gaggaaactg 14760 aaggtcagac tgagtcccatctggataaat gcacaaagct aggacattgt agagcctggc 14820 ttcaaacttc atcccctgacatcagaattt gtgctctctc ctgcctcatt ttacccagcc 14880 ccttgtagct taatgaggagaaattgctgc gctaaatcag aaggcatttt cccaatggcg 14940 agcaagaggg agatgaccaaggttttgcct ttccttaatt ctccataggt ggcaatgaat 15000 agtccaaggt caaggtcagatcccaacact tagacaagaa agcagggcaa ttaccagatt 15060 gggtgagtta gagcagcctaaagacgcacc tgttctcaag tctcctaccc acttctctgc 15120 catagtgaca tgaactcccattctttgatc aacccctttg gcttaaggaa tgtttcttag 15180 acgtcctttt aaaaatatacatatgctact tgtaatgggc tacctatgga tgcagtagcc 15240 ccagctgaca cattatctgggattacttta tcaggtcagt aggcagaggg aagaggacca 15300 gaaggaagtg ggtaagagagaccaggagta ttgcatgcca ctgggaaaaa aaagtgtacc 15360 tgttgtgcat tgtgggtgtggccaccaaga gcagattttg tcccctggcc agtttgccca 15420 agatgtggca ttggcccaagttcctcagtc aggattctag agtaagctct gaaaatcatc 15480 tttgcaagga tgaagccactgacttgagag aaatcttaca tctattccag tcggctcttc 15540 ttccattcta agacctagagtcatttacag atatgtatgg cagagtccaa caacaacaac 15600 aaaaaaaaat tacagactattcactaggca ctataaatgt gagatataac aataaatcta 15660 aagaaattta caatatcgtccatggaattt gatggattcc atcgagtcct cgcaggattc 15720 tagcagcttc attcctccttgaggcccaga caagttaggc tgttataatg ccacatagaa 15780 gactgaaagc agaggtgagaataaagctgc tgctgattga gtttactccc tggtgtgggg 15840 ggtgggggtg gtccagcctcactgctagga ctttctgagt ctttctgaga cacttgccat 15900 ggtcaagggt agcaggatcagggaaggcat tataataaat ataatttgca gagcatctct 15960 ctcctatgca ccagatattgtggtgacact ctgtttaatc cagtatccct actcctttag 16020 atatattgtg attgttttacatgcgaaatc tggcttcaga aaggttaggt gttttgctca 16080 aggtcccatg gaaagtggcagagttggggt ttctgactaa ctccaaaacc ctttaatgtg 16140 tgttcataat taaaaacaataaaaaatgaa gtcaaggtac ttgaaatgca cttttccatg 16200 tggcagctgt ttactgaagcctaccaggtg ccaggccctg ggtcttgtgg gggaagccag 16260 ctgggacctg gcagaacttgcagcctgggg gtctgggaga gtggcagcca ctgggcattt 16320 ggaaggactg tggcctgaaggcaagaatga ggctatggga gctccaagcc aagggacctg 16380 gtatcaggaa ggcactaagcagcgttttgc agcacagcca gggaagaagt tggggttcag 16440 ttcaagtgtg caacatgaggggagaggccc agctaagctc aggcagagaa gggggagaaa 16500 gccatccacc tcttcccctctctcccccgt agacctgttg ctagggcaga ttccagagca 16560 ggtgataggg ggacaccaggctctccaccc agtcaagacc ccaggacctt ctcttggtac 16620 ccacctaccc caggagagtaaggacactgc tggagaaaaa ccatggacta tctcttctgg 16680 ttcctctccc agttaaggtcaccctaaagg atgataagag gcttatctag gcctaacact 16740 cctcagaaag cattttccatctgtatgcca agaattgctc taactagggc gaggcaactt 16800 tcattccaga gtgggagaaaaatgcctctc aaagggaatg ccttgttggt gaacactgta 16860 gagtgaagga atacccagacttcatttcaa agagtggtta tcagatgcac ctagttgaca 16920 agaggtttgt gacatgggggatggtcaatg aagagctgga aaaagaggct ctgtgatatg 16980 gtttggctct gcatccccacccaaatctca ccttgaattg taataatccc catgtgtcaa 17040 gggagagacc cgatgggaggtaattgaatc ttgggggttg tttcccccat gctgttctcg 17100 tgatagtgag tgagtctacgagtctgatgg tttcataagt gtctggcatt tcccctgctg 17160 gcagtcattc tctctcctgctgccctgtga agaggtgcat tctaccatga ttgtaagttt 17220 cctgaggtct ccccagccctgcggaaccgt gagtcaatta aacctctttt ctttataaat 17280 tacccagtct tgggtatttcttcatagcag catgaaaaca aaccaataca ccagggatga 17340 aagacaaaga ccatccatgcatggctctgc tccttcggtt tggtgctgac ttccatcacc 17400 tgggttcaga tgaacaggtggtaacaacta aaaatggtga ccctgaagtc acaaatcaga 17460 tggatactcc cctcacggtgggtgtctcct tcaaaggagc atgtgcacca tcatcttatt 17520 attacacaga catacacagtatgtgtgtgt tttacatata tacagttaac ctccaaacaa 17580 cgtgggggtt aggggcactgacccctccat acaagttgaa aatccatgtg taacttttga 17640 cttcctaaaa tcttaactactaatagccta ctactgatgg gaagccttac tgataacata 17700 acagtcaaca cacattttgtaggctatatg cattatatat ggtattatta caatacagta 17760 aggagataac agaaaatgttatttaaaaaa ccacaaggaa ggccgggcgc ggtggctcac 17820 gcctgtaatc ccagcactttgggaggccga ggcgggtgga tcatgaggtc aggagatcga 17880 gaccatcctg gctaacaaggtgaaaccccg tctctactaa aaatacaaaa aaaaaaatta 17940 gccgggcgcg gtggcgggcgcctgtagtcc cagctactcg ggaggctgag gcaggagaat 18000 ggcgtgaacc cgggaagcggagcttgcagt gagccgagat tgcgccactg cagtccgcag 18060 tcctgcctgg gcgacagagcgagactccgt ctcaaaaaaa aaaaaaaaaa aaaaaaaccc 18120 caaggaagag aaaatagatttactattcat taagtgggag tgggtcatca taagtcttca 18180 tcctcctcat cttcacattgagtaggctga ggaggaagag gaggaggaag aggaggggtt 18240 ggccttattc tctcgggagtgacagaggtg gaagatccac ttatacttat ccatgtataa 18300 gtggatctgt gcagttcaaacttgtgtttt gcaagggtca actgtataca cacacataat 18360 atacaggata cacatataaattttatgtgt atatataata tgtatacata cacttttctc 18420 ctgagatttg aggtccaaaactgaaatacc cacatccaca tagtatttgg cacacaccat 18480 atacccagca aatgatattcctgtgtaaat ccatacctat cttttcattt attttatatt 18540 caccaattgt caagtaatagagcattacag tggagtttag gaaacttgat ttttggttct 18600 gctttctgcc attctgttattttcaactgg cagcttgggt ttcttcatct ctaaaataaa 18660 agggttggat aaaatgatacccgaggtccc tttcagctct aaaagttgat tctttgattc 18720 tcttgtgtac ctcactgtgtgaggtcccat gggaggttcc aaaaggactc tgctctcagg 18780 gagtttacaa tatacttggtgaatggcctt ggagctctca tagcctaggt tgaggtcagc 18840 aaatgtcctc tgtaaagagtcacagggtga atgtcttaga ccttaaaggc tatacagtct 18900 ctgttgcagc tacccagttatgcctttgta gtataaaagc agctgtagac aacatgtaaa 18960 caaatggaca tacctgtgttctagtaacac ctcatctaca aaaacaggtg tgagctgatc 19020 gctgacctag gtacccttccaagaaggaat tgaacgaatg tggcatgatg cctggagctg 19080 ggggcttggc ctttgctgtaaatggttatg aggttggagg agctgccttc agtgagagtg 19140 gccatattca cctgtcactgtccattccat gaagcactga gatctggcaa cctcagggtt 19200 gagctgtaaa atgaagcattctgaattact tggagaacat tctgatgtga atccaggtta 19260 ttaatattcc tatcctgcatgtgttgaaat attcacaggg gctgaagctt aggactttgt 19320 tgccaggtcc atagaaaggtttttgtggtt gcagactcga gtcctcaggt ggaagccctg 19380 gatgtagaca gaactcattccctaattcta ccactacgcc tcctctcact tcccctgcac 19440 ttggggtgct attctgacaaatcattccct tgatatttac tgcttagact cctggctcct 19500 gccaagccat gtgctgctgacacatgctgc tcctctgagc agtgtcctaa catctgtgtg 19560 ctgggacata gccacctgcctatgagaagg tagctaggaa gtttccactt ccctagtggg 19620 aactcacagc aggtctttccaggatgtcac cgtgtgaaaa atataatgat tagctcattt 19680 ccaaaccaat tttgcataccaggtgagtta gccaagccat tcaagcagga aagccattct 19740 gtgaagcccc agacaaccgctcccaaggac agattacctg agaaaggaga atactgtctt 19800 atgaggtcac acattcctaatggtaaacac tcggggcagt ttctgctctt gactccccta 19860 cacccttgat caaggcacttggcctctctg gggaattttt caaccatgaa acagatggaa 19920 ccacattcct gggcctgctgggtcctgggc ttaattcaga ttgtataaac tcatggaatc 19980 tacttatagt ctctggctttggttaactta ctttggttaa ctttcacatt agccactttc 20040 attgtgtgtg tgtgcatgcctaatttacct gatgttgcca ggccttttcc atagtctcaa 20100 atgccatgat ctggcaggaaattggttctt tcttttgcca acaagtaaca ataaagggca 20160 cttttgccca ctattcatatgtttgtattt tgggagtatt tttaactgat tttgttattg 20220 atgtatgtag tagatattcgtcctacttca ttgctaccca gtgccatttg aacacgcttc 20280 ttaggcccag catcttcaaggcagaagcca agaactggct ttcttttttt ttttttttct 20340 tttattatta tactttaagttttagggtac atgtgcacat tgtgtaggtt agttacatat 20400 gtatacatgt gccatgctggtgcgctgcac ccactaactc atcatctagc attaggtata 20460 tctcccaatg ctatccctcccccctccccc caccccacaa cagtccccag agtgtgatgt 20520 tccccttcct gtgtccatgtgatctcattg ttcaattccc acctatgagt gagaatatgc 20580 ggtgtttggt tttttgttcttgcgagagtt tactgagaat gatgatttcc agtttcatcc 20640 atgtccctac aaaggacatgaactcatcat tttttatggc tgcatagtat tccatggtgt 20700 atatgtgcca cattttcttaatccagtcta tcattgttgg acatttggct tggttccaag 20760 tctttgctat tgtgaataatgctgcaataa acatatgtgt gcatgtgtct ttatagcagc 20820 atgatttata gtcttttgggtatataccca gtaatgggat ggctgggtca aatggtattt 20880 ctagttctag atccctgaggaatcaccaca ctgacttcca caatggttga actagtttac 20940 agtcccacca acaatgtaaaagtgttccta tttctccaca tcctctccag cacctgttgt 21000 ttcctgactt tttaatgattgccattctaa ctggtgtgag atggtatctc attgtggttt 21060 tgatttgcat ttctctgatggccagtgatg atgagcattt tttcatgtgt cttttggctg 21120 cataaatgtc ttcttttgagaagtgtctgt tcatatcctt cgcccacttt ttgatggggt 21180 tgtttgtttt tttcctgtaaatttgtttga gttcattgta gattaaaaca agcaatgggg 21240 aaaggattcc ctatttaataaatggtgctg ggaaaactgg ctagccatat gtagaaagct 21300 gaaactggat cccttccctacaccttatac aaaaatcaat tcaagatgga ttaaagactt 21360 aaactttaga cctaaaaccataaaaaccct agaagaaaac ctaggcaata ccattcagga 21420 cataggcatg ggcaaggacttcatgtctaa aacaccaaaa gcaatggcaa caaaagacaa 21480 aattgacaaa tgggatctaattaaactaaa gagcttctgc acagcaaaag aaactaccat 21540 cagagtgaac aggcaacctacaaaatggga gaaaattttt gcaacctact catctgacaa 21600 agggctaata tcaagaactggctttctaac cccacttctc ttttgcttaa tttagctaga 21660 gtgagttccg tggtttgcaaaaaagagccc agatcattat ttctttcatt tctatttttt 21720 tttttttttt agttctggggtacatgtgca ggatgtgcag gtttgttaca taggtaaaca 21780 tgtgccatgg tggtttgctgcacctatcaa cccattacct gggtatgaag cccagcatgc 21840 attagctatt tttcctaatgctctctctcc ccccacccca gccctcaaca gtccccagtg 21900 tgtgtgtgtt gttccacttcctgtgtccat gtgttctcat tattcagctc ccacttatac 21960 gtgagaacat atggcatttggtcttctgtt tctgtgttag cttgctgagg ataatggctt 22020 tgagcttcca tgtccccgcaaaggacatgg tctcattcct ttttatggct gcatagtatt 22080 ccatggtgta tgtgtaccacatttctttat ccagtctcag attattatct ctttagaaaa 22140 aaaataaatg gaattttcttttgaatctta tagatcctat gatattaatt gtaacaccta 22200 atttgagtgc ttctaaatgtctcattagga tatttagcac caaagattta ttttacaact 22260 tcagtacatt atttgggatgcttttaatag agaaaacaga aatgtcaacc cagtattgta 22320 ggtagggctt gtatgaacacagaattatct tgagttgctg ggccagcctt gggaggcctg 22380 cgtaaagcag atcacactgtgagttctttc tcctccctct ccatcccctc ttctgttacc 22440 aaattgaagg cttctcactgagtgaaatca agttataaaa atttctgaaa gcctccttca 22500 agggaaaaga ataatagtaatgacagctaa cattgactaa atgcttacta tgtgtgctct 22560 aatagcttta tgtgaatttattatttaatc cccacaactc tatgtattaa atactattat 22620 taggattgta tagatgaggaaactgaggca ctaatggtta agtggcttgt tcaaggccac 22680 acagcttata catgttcctaacctatcatc tctcctaagg agtggccaaa ggggcctgcc 22740 tggtatctgg ggtgaaggaacagtgttcct ggccaccttg aggttttttt taaaaaaaac 22800 atttctttct ttaattctttcttcccatcc cttttcctgg acactataat aggcttgaat 22860 attgagaata tactaatagtacttgttcac tgtacatttc ccaagtttat atgaaaattt 22920 aaaaaggaaa aggactgttatttataaata tattagttcc ctttaattat tttctggtga 22980 gagagtgaat gggtgagggcaagagcagac ataattgtaa ccaactgtca aggataggaa 23040 gtgtcaagga agcttcagcagtggttggag ggaggtcaaa ggtgtaggca ctgggcaccc 23100 tctgtctctg taggtctgtgggcactgtac acagtggact tcctttactt gcccagattt 23160 atctgcttct ttcaccactcatgagacatg gtgttgggta atacatgccc aattttttat 23220 ttttattttt attttttactttaagtcccg ggatacacgt gcagaacgtg caggtttgtt 23280 ccataggtat acatgtgccatggtggtttg ctgcaactat tgacccatcc tctgaattcc 23340 ctcccctcac ccctcaccctgcaacatgcc ctggtgtgcg ttgttcccct ccctgtgtcc 23400 atgtgttttc gttgtttaactcccacttat gagtgagaac atgtggtgtt tggttttctg 23460 ttcctgtgtt agtttgctgaggacaatggc ttccagcttc atccatgtcc ctgcaaaaga 23520 catgatctca ttcatttttatgactgcata gtattccatg gtgtatatgt accacatttt 23580 ctttatccag tctatcatcaacgggtaaat aaatgcccaa tttaatactc atgacattca 23640 ctacctgagc aacaacatgtaagacagtta actgttatcc ttaccctcag cctgttgaat 23700 tcccatctgg tcagccttcactgaagatgt ggtcttggtg gggatatctg ctgaccatga 23760 gccctgcctt tagtgaaccacatggagaaa taattcaact tatttatgtc aatctcagga 23820 aaaaagctga gggctgattttaagatttta agtggagaag ccaactttag cttaacacat 23880 caaagaactt tcttaaaataaaacttcccc aacagagcaa tggggtatct tgagaaaggt 23940 ggtgagcttc ccatgaacggatgtatttta acagaggctg aatgatttcc gcagggcttg 24000 ctatagaaag aattcatgtatttgggccca acacatagta ctgggcacat agtaagtgta 24060 cactaaccaa tcgcagttgttagcaaatct gttcccactc tagtggccct tgtttttacc 24120 ttctccttag tttacagttttatttaaaaa ggaaaggtga aagtggtcag tacctctgtt 24180 atgtgaaagt ttgtcagggttagaaatgtg attgaatgat gctgtgtttt tgtagtgcta 24240 cagttactca gagtagcaagagaggggggg ttttctctcc catgtcctgt ggttgatgta 24300 actcacacag atttgaggtcctcagatatt tatctggttt atgttgtaac aaaattggat 24360 tgaagatatt tgaaagcagatgcattgatc tttacttcca catttcctac aactcttttt 24420 gtaaagctaa gcatttcgaggtgcaaatga gagtttcaga gcttgattga ttgttgaata 24480 aagtcttgcc taccatagtgatcatattta taaaatatta aacatgctaa tggttccctt 24540 ttgtccaaaa tgtattttgctcagttactt taaaatagat gtgagaactt catagtttgc 24600 aatggcaatg tagacattagaatttcagaa attttcctct tatgaacaat tagatgtttt 24660 ctgctcatat ggccttataaaatctccccc atctctgaag atttggaaaa caggtcatgc 24720 ccagtgtgct tcaggaccaactggagtttt ccagttttct aattgctagc atctgtttcc 24780 tacaagaatc actggaccagaactgctttc ttggttattg atgggaaacc tctcctcaaa 24840 tatctcaaaa tgcttttcagacctctgccc agttagcttg tgtttgtgtg tctgtgtgtg 24900 tgtgcacaca caggcacacatatgcacaca cacacagatg cacatataga ccatgtattc 24960 tcaaggcagg ggggtaattatcaccccaaa agtaataaaa ttggttcata tggggtacaa 25020 aattacactc tttttatataaggcataaat atatacatac agcccttaaa tagatataca 25080 gtgtatctgt ggtattaagtctatataagg ggtgattagg gaaaacatgt ttacaaagcg 25140 tccttgaggg aggtgataatgaaaataggg tggagaaaca ctgatacaga cttacagcag 25200 atatactgga gtgtaaatacacaggtattt ctgcattcac atacaggcca cccatgcata 25260 tgatgcatat gtatctgttaggcataaaca cacgctatgt agtttaccca gggaattgaa 25320 ttcacagtgg ggcaggactgaattctgtgg tctttctaaa attctcttca ataaagaatc 25380 tctccgtgaa ggatttgttttcactttcag gatccccatt aaaatatttt tttaaaataa 25440 acggtcattc ttattttgacttgcaggcac ttcaattaac atcacacagt cttcaaacac 25500 agagagtaca cattcgctgtcattctctgg aaaggctgcc tgttagaaat gagctgggtc 25560 aacaattcat ttcactagacatctgctgag catctcctac gtgtggggca tcacagtgta 25620 tcccacagga tgtgcaggaatgcaggacaa ggtccccaag gcagagtcag atttagaggt 25680 gggctttata ggtgtgagcacctgatacca ttgaagatgg taggttagaa tggcctgaga 25740 gtggggaagg ggcttgcctcacagcctctg gaactcttga tacaactttc tcttccatcg 25800 ctgcccttta tcaaccttattctaatgccc atctttcctc ttgttaatta tttttgctat 25860 gatcgtaagt tgttttgatttcgagtgccc ctaaagtctt ttaggaagta gatggtatgc 25920 acaaatccac aacataacaattaaagtcta attttttaaa aatcttgtta tggattcaca 25980 cagtttcaac caatagctgtgtttgacaag gctgtcaagt atcagtgttg agctcctcac 26040 attctgtctc attaacctctctttctacta tagcaaccct ctgctcacac atacccactc 26100 ccagatgtat ctgaggaagctttcttttcc ttccctcatc cctacattca tcagttctgt 26160 agatgggacg tgaggaaaccccatcttcct ccaatacaag cctgtctcta ggaaatgatt 26220 tccagaaata ggggtatattagtttgcttt gctgtgtaac aacttaccac aaatttagca 26280 gtttaaaaca aatacctttcttatctcaca gtttctacgt gtcaggaatc tgacaatggc 26340 ttagctgggt cctctgctctgtatctcaca ggctacagtc aaggtgtcag ctaggactgc 26400 atccttgtct gtagttctgagtcttcttcc aagctctggt gggtgttggt agaattcagt 26460 ttcttgcagt tttaggattgaggccctcag cccctagagg ctgcctatgg tacagtgcca 26520 actggccctc ttcacaggaaattcacatga tagtagcttg cttttttagg actagcaaga 26580 atctcttttc tagcagctttcacccagtaa ggccacccag gatagcatct cttttgctta 26640 attcaaaagc aactgatttaggccttgata acatctgcag aaaatctttc ctttactatg 26700 ttctgttagc tagaaacaagccatagatcc cattcacact cgaagggatt atatataagg 26760 catgaatgcc agaaggcaaggggatctggg ggtcacttta gggtctgtct gctgcaggtg 26820 gcttgagcta atgttcctccacaataataa cagtagccag atgcttcatc taatcctcac 26880 atggacagct tgaggagagacttgatatgg gtctatttca catgaaagga acctgagttt 26940 cagagatcca gtcccttttccaagatcaca gagctggtag gagaagaacc cggtcttgaa 27000 tccaggtttg tctgacatcacaccaatcaa acaacaacct gtgtgccact taagggagct 27060 gacagtctga aatactccaaggttatcatt atattgcaaa ctcaagtttt attttgacct 27120 ttctggatgc tcaccaactttgtttaatct tcctaaattt agattaagag cacatttctg 27180 atcttggact tggcatcagtatattttgta aggcaggatg aacaccctta ctatgtaaca 27240 ggggttttca gaagaaactgcagtgtgaat gttattattt aaatttttgc tgtgaggtaa 27300 ctcacctgca ggcaggctcagagagcttgt gtgatgctac acatttatgg ttgatgggag 27360 tgagatgtga aacccaccatccttctatca cacttgggtg cttgttggcc aactcaagtg 27420 gttttctaaa acaaaaaggaaacaaagagt aaaagtcaag atggaggatt ctttgtttcg 27480 ttcaggcagt tatatttttagctgccccta actcactgac ctattgctgg aatggaaact 27540 agtgacttaa acatgaatggaaaccacttt aaaaagtaat agggcattgg tgctgcctac 27600 agtgaatcag agggtgaagcattcagatga gtgcaaaaag tctaccttgt tttgtttgat 27660 cttaacaata cctttcatttgtaaaacaca cagtagccta aaaagaatat ttgaataatt 27720 atataattta attgtcctaacaaccctgtt cacatgaccc aactttccag atgaggagaa 27780 gagtttcagg gagaaactccatgtcttccc caaggttgcc tggttcagag gaaaagcagg 27840 gtcttgtgca tagggattcacttccaagct cctattctac acttccctct caagaacaaa 27900 gatgcatata tggatggagacgtcgctgga gagagtgcat ggatgtgaga tgaatcccat 27960 catagataca tagaaacacccaggagttag acccagagtt tagccatcaa tgtcctgatg 28020 gtgaagttcc tcaaattgctagcccagggg tggggttttc actattgcct cctattctaa 28080 agggcagata ttattttccctgcaactctg cccattgggg aattttggta tctttaatgg 28140 gatgcctacc ttgccccagctaacttctgg taaattttaa tattccctat ttacaaaaca 28200 gaaccactgc agagattgctataaagagca aggtgggcct ttctcacttg accattcaaa 28260 ttaaactata ttacctgacttacgtgctta ttaacgtcat gagtacgatg ttgtaagggc 28320 agcactggag atactggtcaaacagtaata ttatcaccat agaaaactgg tgaatctaga 28380 aaactacttc cattgatctacatttcaatt tggggatttt tcttaatgtt tttaccaaaa 28440 tttcaaagca attctgacttttcctagcca aaatgatatc acctttttta cagagataat 28500 atcaggaatg agaatatttatagagctcta cagcttccac aggatgctga ggctaattgt 28560 cagagaagct ggaaaagggtgtctcaggtc ttagtgatga caatcaggaa aggagacaat 28620 gttctcctaa aacaccacctggaaagcatc agtgggggtg gggagagtac ttaatgtcag 28680 agtcaatcct gttgtgtgtctcttcctcct cccaacattt tgacaagagt gaaacaagat 28740 tcaaatggat gtcaaatgcagcctagaaag gcagacagaa tcctgcacat cagcacttcc 28800 aggctacaga gcagttctagccatgggggc agaagagtca tgctgctacc tgggaagggg 28860 agagccttgg accctaggtactgaccctct gtattcagaa cccaagcctc atagactgag 28920 actggccttt agctttctccccaggtcatt ccccctctgc cagtggctgc agctgcactg 28980 agcctgccag aggtcggagtctggcagaaa ccccatttgt cttgacctgg aaaggatctg 29040 ccctcctgtg ctcacactattggctgggcc agcaccacaa agcgcaaggg aggaacttga 29100 gaagtcagca gcagcctttgttctcaaaat gcttatgctg tgatttcata ctctttctaa 29160 ttttaatcta aacttaacctttgaaggcaa tttaatagaa ccctgctaaa acaaggataa 29220 tagttaagcg tggcatgaatctcctcaggt tttttaatgg caagtagcca cagggattca 29280 tgctttggag agatggctggatcagatgat tcctaaagcc ccttgtgatg gttaattcat 29340 gtcaacttga ctgggccataggaggcccag atagttgatc aaacattatc ctgagtgtat 29400 ctgtgagctt aacatttgaattagtggact gagaaaaaat agattgccct ccctaacgtg 29460 gctgagactt aatcaattgaaaacctgact tggcagagtc atgggggcag aactgccacc 29520 ccagcaggct aagaaggtgggatatcaaac taaaaaggac tattctcaga ccttaagatc 29580 taataaaatt tgccttgctaggttttggac ttgcttggga cccatcaccc ctctcttctt 29640 tccaatttct cctttttgtaatgtttatcc tatgcctatc ccaacattac attttggaag 29700 catgtaatgt gtctgatttcacaggttcac agctgcgaag gaattttgcc tcaggttgag 29760 tcacacctca ggcctcacccttacctgata tagatgatat ttagatggga ctttggactt 29820 tagactttag aattgacactggaatgagtt aagactttta gggctgttgg ggtggaataa 29880 atgcatattg catgtgagaaggccatgaac tttggggggc cagggacaga atgcagttga 29940 ctaaattgtg ctcaccaattcatatgttga aatcctaatc ctcaatgtga ctgtatctgg 30000 agatagtgct tttaggaaataattaaggtt aaatgaagtc ataagagtgt agccctaatc 30060 caataggatt gggggatttataacaagagg aagagagata ttctctctct ctctccctct 30120 ccttctccct ctgtctaccaagtgaggaca gagccggaag gcagccatct gcacaccagg 30180 aagaaggccc ccacagcaatcaaattagcc ggcatcttga tcttggattt cccagcttcc 30240 agaactgtga aaaataattattttgttgtt atttaagcca cgcaatctat aggattttgt 30300 tatggcaggc tgaacaggctaagactttgg tgttgtgatt ataacaacga tacaaaaatc 30360 tatcttatag ttccctcacaggtcaaccct gactgtcagc taacaaatca ataaacattt 30420 tctggatttc atctgtctataaggcactgt agagaatata gacatgtgta caacatgttt 30480 ctttctccaa tggagaccacagtttagcag aagagataga gcttaggtgt gcacaccaac 30540 tggaatctaa ctttaaagggcagcagaaaa gcatgctgaa tgttgagttc agagggaggg 30600 gccataactt ctggcaggacaattatcagg aaaggttttg tagagtgaga agtgatgtta 30660 ataagtgtct ctaggaagcaaacattttgc agagcttccc acataccaac taagttaatg 30720 gggtcaaatg cttgggttaagtccacagtg ccactcatgt tgctattctc agcatcttcc 30780 agtggctgcc agtgaaagagtatcaggtct ccagttccat gggcaacaca agaggcacac 30840 tgactttcaa gtgagacacactgccactga tacatgtgag tagttcatgg aaaatgactc 30900 aggttaatgc tatcctggaattgtgtacaa ataatatgac cccagggtct tctgtccctg 30960 cacaagactc ctttaagttgggcaaggtca catttactgc ctctttataa tcaaagcaat 31020 taagtgaccc cacaagtggtttagctacta gctgctgtac ttgtggaatg cagggtctta 31080 ccttaccagg gtctccacccagtgaatgag cacagaggaa atctcagctc tgaaaaccaa 31140 acaggacttc ataacttcattgagcccagt tgttttcaaa tgtttcctgg tggcaaggaa 31200 ttctttgtta tgtcaactaaatcatgtgtg gagcctcact atagaaaaaa aagtttaaag 31260 aaggccttct ctggcttatcctggctgtgt ccttcttgac cccaaagtgg cccttaaggc 31320 accctcacaa aaagctggagcagacaattc taccctttcc atttgcagac atcactacct 31380 tcttaggcta caatgtcctttcatcagaat gaatgcatgg caggattttt acctgcctga 31440 ggtcacacag ctaatcattggccaagctgg gagtagaagc tgggtttcct gactcctaac 31500 ctagtgctcc tcaccatctgggtagcttta ttcaatggta ccaaggattc tgaccaccct 31560 gcattcctgt tacaccaaagaatccagtga tgctggggaa atcaatgatg gtgtgtcagg 31620 atctcagggc atgatcagggttaaaggcac tgtgttagtc tgttctcatg ctgctaataa 31680 agacataccc aagactgagtaatttataaa ggaaagaggt ttaattgact cacagttcca 31740 catggctggg gaggcctcacaatcatggtg gaaggtgaat gaggagcatg gtcatgtctt 31800 acatggtggc aggcaagacagagcttgtgt aggggaactc ccctctataa aactatcaga 31860 tatcctaaga cttatttactagcacgggaa agacctaccc ccatgattca attacctccc 31920 actgggtccc tgccatgacaggtgggaatt atgagagcta caattcaaga tgagatttgg 31980 gtgggtacac agccaaaccacatcaggcac caacaagaga taattgaggc ctatacatgg 32040 gagaacaact acaaggaccctttacttaga tctgaccctt tacccaaaaa gagtcactgg 32100 aaggaggatg acctttggaaaagggatgca gccaacccag agtggcctgg tagggatggg 32160 gccaaggcaa taaatacccaacttcgcgtg cattaggact tctgatccta atgcagccag 32220 agggcaggga gccccttgatgctattccca ctgtcagcct ccaggcccca agcagagtgg 32280 tgaggctaga gagcaggtctggagggacac agaggcattt ccaacccagc tgctaagaaa 32340 ggagtcactt cactgttgcactgcatcgag cttattgaaa cacagtgttt tggggatgag 32400 cctgcttttt acagaaaggtacctatggga agaaattaac aaggaggtca aatacacata 32460 cttcctgctc tgctgtactaaatggcacag caacttttag attctcgcct gaaaattaga 32520 gtatatgaga ttaaaccctgaaggtagaat gaattgcttc atgatctgct ccctgttatc 32580 tctctgggcc catctcttaccactccctac ctcactgccc aagtgacaac cacactgaac 32640 aacagctcag ggctcccagaggggccaggc tctttcttct ctcccacagg tttccctctc 32700 tatgtggatg ttccttaccccctttgccag ggcttagctt ctagaaggcc tttccaggcc 32760 ctccctccag catgaccaagctaatttcag tgtctctctt gggtgttccc acatctccca 32820 tgcacttttt cactgctcagatcccattgc cttataagag tatgagtgtg ccaaatgttc 32880 tgtgtcttcc ccgtgaggcagctatgacgt caaggatggc agttaccgac cttgtttccc 32940 tggtattaag acagttccaggcatagagct gctcgatgaa tgtctttgga gggaaagaag 33000 gatttcttga gacccagtcatctcatcttt aaaaagaaag aaaacatcat ggaaacctca 33060 atgaattctt cattagtgattttgaatgga tggctgtaaa caaagctgct cgctgaatgg 33120 cttatgtttt tcctttaaagtccaccatca gaactccatt ttgtgggtaa ctagccccag 33180 cgaaggcaag aagaccatgaaagccttgcc ctgcagtgac caggggagag catcctggtg 33240 acctgtcatt tgcaaacatctgtatgttgt ttcaaagttt atgaagcaca cgtactacat 33300 tacctagaac ctcaaaacaaaactgtggag gcggggcact tagtacaagt gtcattctca 33360 ttttactggt agagaaaagctgaagctccc aaagtcaact gtctcgtctc aaagccacac 33420 agccgggtag aagtcaatgtcaagctgggt tccggagcac ctgtcaagtg tcccttccac 33480 cccaacatgg tgagtccagggatagcacag gtgacagtac tggcattgcg gacctgaaga 33540 cagcagactc ccaggccaggaggcctggaa agtactcgag aattaattct acacatccct 33600 ctcatcttga gcacaaggaaccaatgcact gatggctcct ctaatgtttt atttgtcttt 33660 taatcccaag tacttttttaaatctctagg ggaaaaccaa ttcaattttt gtactagttt 33720 tctctttcct ttactatgttccactaagtg ctttcccaca ctctctttga ctcttttctt 33780 gaagggccta ggttctattcatctctgtga tgatgttttc tttcaaaaaa tggtttttct 33840 gaggccctac tatgtgctggacattgattt tagtgctggg actacagcag tgaacaaaag 33900 agatatggtt cctgacttaaggagtcttgt gttctagtgg atgagacaga gaagatataa 33960 agaaataagt caagtaatttcaggtgataa aaagtgctat taaggaaata actggaggac 34020 taatttaaat gggagaagggtataaaagac ttatctgaga aaggcattat atttcaggcc 34080 cgaaggcagg ttcttagcccttaccaggtg aggaaatatt tggtgattga atggatgtgt 34140 gcatacattt aaatgaatgaatgaatgaag ggttgtctat attctctctt tacacaggca 34200 ttcccccaac ctaccttgtcctacctgttg cctacatgcc agccagtcca tacctgaatc 34260 ttctatgtgt cttctcagagatcccaagcg tctgtaactc ctaaggccgt gatatgcagg 34320 agcagggtag ccagagcggagctataagga atgccacaat cgctcttcca accgattgtt 34380 acaaaactct tctcaatggtgtgtcttgtg ccaagcattc ctcttcctgt cctgagggct 34440 gtatattgac atcacttacccaaacccaac tcccactagg tagtataggg acactactca 34500 tttgatgaca ctttggtggctctgtgcatg acagcacaca ccctgtccca gaatatatgc 34560 atttttatgg cttcctagtaagaatactat ccaagtcctc cccagcattc ctctcaccct 34620 ccctccctgt gctgctgcatgaactttaga tcaggtgagg tcaggcagtg aaaaagtccc 34680 tgggcagagg tctgaggatttaacagattt gttgagcatt atgatctctc actgaatgta 34740 cgttacaagg aagcccacctcctttgtttt attggcattt cattttcttc catttcccag 34800 accttgtggc cttttccctgtgcagctcat aaaagtaggc aaccagcctt agtaattcac 34860 cttatgcttg aatcgtttctcaccttttac caaggcttat tccatttcta gatttctcat 34920 tattcttttc agctcttacaaactccaaat tcatgccatt tagactgatt cccagaaaag 34980 agataaaagt gtcagtcagtactaaaagcg aatcattgac ttcttaaatg tcccttatta 35040 aaaccactca tctatgcagccatgcaatct gagacaaaca gagttgaccc aagtctttat 35100 ctccatgtgt aaaatgtctcatgagttgca gagtcacata ccacataata acgtttcagc 35160 caacgacgga ccccgtagaagacggtggtc ccataaaatt ataatactgt atttttatta 35220 tgctttttct atattttgatacacaaacac ttacccttgt gttacagttg cctacggtgt 35280 tcaatatagc aacatgctggacaggtttat agcctaggaa caatagacta cactatataa 35340 cctaggtgtg tagtaggctggaactatcta ggtttctgta agtacactgt atgatgtttg 35400 ctcagtgaca aaatcacctaaagatccctt tctcagaatg catccctatt attaagcaat 35460 tcgtgactgt ataaaaaaatttataatatc atgaactcat ccactgaaag gactggaaat 35520 gccttagaga tgaacaagcttgattcatga ggacactgag tcccaagcag gagcagcaag 35580 gacaggcccc aggtgacccaaactgataag atggagtcag gacaagaatc acagcccctg 35640 catggttagg tgtccaaggtttgttcacct ttccaaacta caggttctac agctacctct 35700 tcttacacag aaaccttcccctaaagctga gctccctcac atttcccgtt tctcatattg 35760 tctttgccat cttccaacattccaagatca aaacctcagt gtcatccttg actgtcccct 35820 atatttcttt ctttttttcttttttttttt tgagatggag tctccctcta tcacccaggc 35880 tggagtacaa tgctgcaatctcagctcact gcaacctctg cctcccaggt tcaagagatt 35940 ctcctgcctc agcctcccgagtaactggga ttacaggcac ctgccatcat gcccggataa 36000 ttttttgtat ttttgtagagatggggtttc atcatgttgg ccatgctgat cttgatcctc 36060 aggtgatcca ccctccttggcctcccaaaa tgctgggatt attggcatga gccaccgcac 36120 ctggcctgtc ccctgtatttcatcagacag agagtcttcc tccagagtgg ttcccttggt 36180 agatgcccac caaaactgaacagcaatatg aaagtgcaag gttttcctcc cacctaggcc 36240 tggcccccac agaggtggtaactaagggag tccacattgt atctcattga gtatcaactg 36300 cttacagaaa aggaatgtaatatccattat ggtctttgat aaatatataa atcagatttg 36360 cccactgcaa agcacaagccttgagaaacc acattacttt ttgaggtata tttagaagct 36420 agttgtttaa ttttcttggaacttacatat cacttacttc tggctcctta tttagtttgt 36480 aaaaagttca ggatgtttcaaaatttcaga aattatataa gtagatacta ctcatctagt 36540 aagcacgtgt aaaatgaaatagcaataaga cagaaaatga ggccaggtgc ggtggctcat 36600 gcctgtaatt ccaacactttgggaggctga ggtgggtgga ttgcttgagc tcaggtttca 36660 agaccagcct gggaaacatggaaaaaccac atctctacaa gaaatacaaa aattagccag 36720 acctggtggt aaatattgtagtttcagcta ctcaggaggc tgaggtggga agatcgcttg 36780 agccctggag gttgaggctgcagtgagccg agatcacacc actgcacttc agcctgggct 36840 acagagagag atcttgtctcaaaacaaaac aaaacagaaa atgaaaataa gcactaaatg 36900 aacctgtaat aagggtcaatgcatgcttca aataatgatc tttgagcttc taagaaggca 36960 aggagggaaa aaagacacaatgagttatag aattctcttt ttgagagtgg aagaagacaa 37020 tttcttggag aagatgaaattccctgtcat tgaattactt tgaaaaatat ttttttctat 37080 ttataaaagt aattagataacttctgataa tgttttggtt tctttctagg tttttttttt 37140 ttaattattg gaattatacaaccgatacat tttgcagcat gctctattca caacatttta 37200 tcactagcat tttcccttaacattaagtat tcttttcttt gtggtgatag attctaaagc 37260 catctctctc ctttttcaacttttattttt gttttggggg tacatgtgct ggtttgttac 37320 atgggtaaat tccatgtcgctgaggtttgg tatataaatg atcaccgtca cccaggtagt 37380 gagcatagta cacaataggtagttttccaa tgctcacccc actcccactc taccccctct 37440 agtagtccct agtgtctattgttcccatct ttatggccat gtgtattcaa tgtttagttc 37500 ccacttataa gtaagaacatgcagtatttg gttttatgtt cctgccttta tttgctacct 37560 gaaacagcat ggtactggcataaaaacaga cacatagacc agtgaaacag gataaagaac 37620 ccagaaataa agccccatgcctaaggccag atcttcagca aggttgacaa taacaagcaa 37680 tggagaaagg actccctgttcaataaacag tgctgggata acaggctggc catatgcaga 37740 agattgaaac tcaacccctacctttcacca catacaaaaa ttaactcaag atggattaaa 37800 gacttaactg ttagacctaaaactataaaa ttcctagaag aaaacctagg aaatgccatt 37860 ctagacatca accttggcaaaaaaaaaaaa aaaaaaaagt gactaagtct ccaaaagcaa 37920 ttgcaacaga aataaaaaattgataaatgg aacctggtta aactaaaaga acctctgcat 37980 agcaaaagaa actatcaacagagagagtaa acagacaacc tacagaacgg gagaaaatat 38040 ttgcatacta tgatgcatctcacaaaggtc taaaaggcca tttctcatgt aatggtttat 38100 aagtagtgtg tggggatcactcaaaaatat aatgaacaat gccatcaacg ttttttaaat 38160 agaaaatgca gtagaaattttgggtgatta tttcatctag taactgtaaa aactgaggcc 38220 agggcattaa aatgtgattcaaggagaatg atttttcaca ggtgctaatc ttatgcagta 38280 ggaatatcta atcttgtcagcccaatgtga ttcaggacaa ggcagaaggc tggaagagtg 38340 ggtggagccc acatagccttagtggacctt ccctaaatgt cacttcccag agacaggctc 38400 ttaattcaaa gctgctttaaggttttgagt taccagggaa tctattcagc tagttcagtc 38460 tttaagatat tgaacaatgccttagtgaat gaggaaagaa cgtgacatgc ttagagggtc 38520 tcttgtctat tcaaggagttactttggact ggagaaaggg gaggaaatgc cccttcccct 38580 gaactttgaa gattggtcttcagttagcac atataataca tgtgtcctgg gtcttcatgg 38640 cactgtgaga tataaagggaagattcttat tctaacattt ttgaataagg aaaacaaagc 38700 tcagaggagg taaatgacttgtccaaggtt atatcccagt gaataatgga accagggctc 38760 cacttagtgt tgtctaacctggtccgtggc tgttgtgtat cccaccctgt tgtgcatttt 38820 ttttcaaact ctggctgttgagctcctttg gaaggctgat ccacagctct ctcataatta 38880 ttttctgttt tttttttttttaatggagtt ttgctcttgt tgcccaggct ggagtgcaat 38940 ggtgcaatct tggctcactgcaacctccgc atcctgggtt caagccattc tcctacctca 39000 gcctcccaag tagctgggattaaaggcatg tgccaccatg cccggctaat gtttgtattt 39060 ttagtagaga tgggatttcactatgtgggt cagctggact cctgacttca ggtgatccac 39120 tcgcctcagc ctcccaaagtactgggatta caggcatgag ccactgcgcc cagcctcata 39180 attattttca atcccaacttctagtacaga tgttctccaa gtgtggaccc tacactagaa 39240 gcctcagcat cacttgtaaaagtgttagac atgcaaattc ttgggcccca ccctggacct 39300 actgtgtttt aacaaggctttcaaatggtt ctgatgcatg ctaacattgg agaaccactg 39360 ttctagaagt tcttaataaatattgttgaa tacttcctag aatccccata gaccttcact 39420 gaaatattta aaatatttaagaatccccac atatattttt attggaaagc ttacgattcc 39480 tgctcaggaa tcactcattgttagccatcc ccagtcaaag agaagagtat tttatcacac 39540 aatgacaagg aagcccaacactaagttctc ggatatggaa agcactgagg ggaaaaaagg 39600 caccatagat gggtcagaccaggtataaga tcataatgta atgctgttta ttgagcagtt 39660 attttgcacc acaaactatccctaaatgct ttacatggac aataaattca caccacaact 39720 ttttaagtac cacaccatatttctacttta ctgaggagca aactgaagcc aggaaacttt 39780 cccaagatta caaccctagtaagtggcaaa gcctagtcag acccaaagcc caggagcttc 39840 cctgctattg tcttttgtcttcttgctctg ttgcttccag agaaaaaccc caccactgca 39900 gcaagctaca gggaaagatgctgagtggaa acccactggg catcccttgc acctagaaca 39960 gcaccccaca cattgttgatgtttagtatt tgtcaagtga atgaatgaaa tgaactatac 40020 acaacataat tctaatagaagtagtctgaa gaggatttcc agggaagatg gggtctgagt 40080 tagtatttga aagtagagacagataatgct gtccaaaaga gagaaatgac atgaacaaaa 40140 gtgcagacat agccaacctgagcctagtca gaggaggtgg gcaagctgtt tgcctagagg 40200 ggaggggagg caggggaagaggtcttgaaa ggggagaatt tataaccctg gaaaaagaaa 40260 gggtccctga gaggcagctcaaggagtttg aattttatcc tattattagt gcaaagtcat 40320 tggagggttc tgagtatgggctcaaaaggg ccaaagaaat aatttaggat attgtatcat 40380 atccattttt ttatttttaatttacaaaag atggagggaa ggtagctggg ctccagatct 40440 ggggggtttg ttgaaatagcaactaaaaga tcatctttca aaaaaagcca cataccattt 40500 tgattcatta caatttgaatgtagcaagaa taattacaat aagcttgtca ttaaagcttg 40560 cactggaatt tgataaaaagaggtttatgt ttcactataa acacctgaag tggagtcata 40620 ggaaaaagca tgcaaaatccaaaaggccac aaagtcaaaa ataaagaatg aaaaagttac 40680 tataatcctt gttattcaagggtgaaataa aaataatcac ccaatctgtt tatttaaaat 40740 aaatatttat tacaaatatttattataaat caactatata gtttaaacac cactttcttg 40800 caaaagacaa agccctgaaaggaaataatt tattcttttg atatgtgggc agatctgtat 40860 gaactacatt gctcatttagcaatgaaaag tcaaccaaac atgggaagac aatatgcaca 40920 ttgcatacat acagcacacacgtaacatcc agccctaaaa agcatcagca atatcccttt 40980 tgttgttgtt agtgttattttccgacaagc tgccttttgt ctattctagt taatgttagg 41040 aaacctcata ggtctatgccattataatgt ccattaccca tttgaatatt tagacagagt 41100 ctggagtgat tattaatactgagaatcact atgtttgatc cagtgaagga caaaactgta 41160 gtacgatgaa ccaaaagtactaagaagggg ttatttgaca tttttaactg ctttgaattg 41220 aataaagtct gtctgtgcatgggcattgta tgaatgagtt tgccattaaa agtaatggca 41280 aaaacagcaa ttacgtttgcaccaaccttt attaaatgta agcttctatg tctaaccatg 41340 ccttcaaatt cttttctgaaagtagaaatg catgtacata gagtgaccaa ctcatcccag 41400 ttttccaggg accttcccagttttaaatcc tgaaagtcct ggaattcccc cagtcctagg 41460 caagctggga tgtctactcacactacacat ttaacaaaga aagaaacaag cagacaaaaa 41520 acaaatctac caagaaaataataaaaatga aaggacactt ttccgatgtc ccacccagta 41580 gtaaacgctg cttcccaagacatgaaaaac atattgttga gtctcctatt taccatttat 41640 tccattctgc cgtatacgtaatataattat tatattaacc ttcatctctc cataggtgac 41700 tggaagctat aatcacttccactgatcaga gcccttctat gcacatggtc tcatttgacc 41760 cttgctgtca ctctttgtgataggcagaga aggtgtcaga ggcccagaga tgagcagccc 41820 ccacttctta ggggagcatcagcacaggaa cccaggattt ctgattccca gtctggtggt 41880 tttttttcca tcactgatcatttacattct ctttatgaca ttcaggccct ggtcctttac 41940 ctatataaca tagagaaataggctatttat aatttacaac ataaattaag ccaaagactt 42000 ttaagcactt tttaattatattgtatacct ttcaagtgag ggtgatgaat ggtactgtac 42060 ccctatttca tagtaagagaaatgaaggct ccagctaact tggctcaagt tgtacagcag 42120 ttgcacctta gagattgacaggtaacatat tgtcttgttc tttgtaatct gatatacttg 42180 gatttaaagc acatttcaagatttttttta agattagcaa cataactata tacatatata 42240 tttaaaataa atgtccatattgatagagac ctagacagaa acaagcactt gcctgcccca 42300 atgagaatct gagggcaccactggttcctg gcacactgga ctgaggatgc catctataca 42360 tgtgttggac agtcactgttggccaaagtt aaaatataca ttttaacttt gacagaaata 42420 cttttgtttc agttgcatatcgaatgacag gagccacgtg ggcatcactt taggatctgg 42480 ttactccttg gcacctaagttactttctat ttcattaaaa gtctttcttt catctttatt 42540 tgccattctc ctgtatctgctacatggcag gctagtgggc ttgaatttgc tggggtttcc 42600 agtccccagg tcaggccgacccctgagtgt gcctcaccaa agctacatcc tgctcactct 42660 ccagttctaa atcagagcctttggaggcac aagtaaaggg acccaagggc agatcagagg 42720 ggttttcctc ttcatatattctttcttttt ttgtcacctc ccttttagct tactttattt 42780 gttttaaaaa tacaagatgactgtgacaac tcaaacctta aagaaatata taggtctaca 42840 attctttatc tgtaatcctgaaattcaaaa agctctgaaa actgtaagtt ctttcataag 42900 tttgtggaaa ttcactttgaggcaaaactt ccttgaactt tagagatgat ttgtagtcca 42960 tctttgcccc acttggtgctgtggagatgt gattataaca gacttgatta cagcagtgtg 43020 gtgtcccaga gcctgcagggggtggggatg gggttacata tatatccaca catgttacct 43080 ttctacattc caaaatattttgaattttga aacacatctg ttcccaaggg tttcaaatat 43140 ttctttgctt gaaaccacataaagaaaata tgaagactct cctttcacac accccttgaa 43200 tgcacccacc tgaagaaactgatgttatca gtatcaggtc ctgccctcaa ctcctctcta 43260 tggcatgccc tctctctctctctttctctc tctctctctc tctctctctc tctctatata 43320 tatatatata tatatatatatatatatata tacacacaca cacacagtag taaacactgc 43380 ttcccaagac atgaaaaacatattgttgag tctcctattt accatttatt tcattctgcc 43440 ctatatataa atataattgttatattaacc ttcatctttc cataggtgcc tggaagctat 43500 aatcacttcc cactggtcagagcccttata tgcacatggt ctcatttgac tcttgctgtc 43560 actctgactg ataggcagagaaggtgtcag aggcccagag atgagcagcc cccacttctc 43620 aggggagcat aaggacagaaacccaggatt tctgattccc agcctggtgg ttttatatat 43680 aaaactatat atatatctcctctctctcac acgtacaata cgaacacatg caagattttg 43740 tttttaaata caaaaagggatcacactgtt cttattactt tgtcatttgc ctttttctcc 43800 tactgtgaca ctccctctagaataatacat aagaatcaaa ctcatgcttt taaatagctg 43860 tatactattc tatcatatggttatatcaca ttgtaatcaa ctattctgct tttttttttt 43920 ttgagatgga gtcttgctctgttgccaggc tggagtgcag tggcgccatc tcggctcact 43980 gcaacctctg cctcctgggttcaagtgatt ctcctgcctc agcctcccaa gtagctgaga 44040 ctacaggcat gtgccaccacccccagctaa ttttttagta gagatggggt ttaaccatgt 44100 tgtccaggat ggtctcaatctcttgatctc atgatccgcc cgcctcggcc tcccaaagtg 44160 ctgggattac aggcatgggccactgtgccc ggccacctat tctgctattg atgaacatgt 44220 tttcatagta ttttgcttttgaggatcaaa tattagtgat tatattgtaa aaggaatata 44280 tatctacagg gtttaattcattttatatat attgggatgt tggttggcaa aaacaaacaa 44340 acaaacaaaa acacccacaaaaaaacacat ttctctctgt catacaaata gaaaccctga 44400 gacaaagggt gttcattttaaaaaatgaaa agaaagagtt agctgcaagc gagttatttt 44460 tcctgcaaca ggcaggctttctatgaacta gaggttgcac agaggcagac tgctaagcca 44520 ctgaactgct cagccatgggcatcaggagc tctcaggaca gcacctttag agagcccagt 44580 ttactaaccc agccatgtcatgtttccata acttaacccc tggaggtagg gtccgacatc 44640 tgactggtcc cacaggcaaagaggcaggta acttcagggg gtgcaggatg gggcctgttc 44700 gagatgaaca tgctcagtgactctaggttt ggttggcttc taacagacaa gccaagtcat 44760 ggaatttcaa taaataatttaacctcagct ccagtttcag cttcagtgtc cactttcagc 44820 acctggctgc cattgttatttgcaagcagg ctcctcctct gcacgaggta tggaaatttg 44880 gcagagattc actgacatttttagaaccca gtagggccag attactatga acaagagtgg 44940 agttttgttt ttgtttttctttttgttttt cctaaccaag aattatgtga aatctggatg 45000 tgcaaagatg cactttttaaatgaaaaaga aaagctggct atcactgacc caatgcatca 45060 gttttaaagg tcttccgattgttgctgtat gaggccttta tcttgcaaag gttctgaaga 45120 ccagccttct caaacactaagaggcataaa tcacttagag gtcttactga aaatgcaggt 45180 tctgattcag taagtatggggctggcctga gaatcttcat ttctaatgag ctcctaaagg 45240 atgctgatgt tgctggtctgtggacctact ttgagcagca aggctgcaga caacccagaa 45300 tttcctccta acaagaaaactatccctctt agggcctttt ctgggagttc caggggagag 45360 ataaaaagct cttaatgagacttgattgag agataaactt acaagaggac cctggagctg 45420 taggacattg tgttctagctggaatttcaa tccactaaca aagaagacag gatatggcag 45480 tggtaggttc cattattgtatgtcatcctt tcagagcacc tttgatcctc acagagaact 45540 cgtgaagtag gtgggcagctgttattattc ccaattacag ggatggagac agctttgagg 45600 ccaattcaag ttatctttgttttttaaatt attttctggt tgaaaagata gagagaaaac 45660 acatcttgct gaaagctaatcacataattg caagcgctat acagtaaaag tccttgatac 45720 ccatactcta cttatgttaaatttgtttct aagccttgtt tctgttctgc cctttggtat 45780 gtctctgaga cactggtcttttcatgcagg ctacattttg aacaactaga gtaaatgtgt 45840 ctggccctga actgtatcataggactgaga agagaaaagt aaaacaggat ctgttttaca 45900 acttagtaga agaaactcatgttacagaaa ggtccataag agctaactag gaggacatag 45960 gggaaaatta tgcttctcattttagctttg ttcttatctt ttaattcttc agcaggcctc 46020 ttctctgtgt agaagagtcagcagaattgc atctaaggtt gactttataa ataaaacatg 46080 tcatcaccaa ataatgcttacattctgtcc tacgaatcct agttgagacc tcagaatttt 46140 ctatattatg tttccagcagcctccttcaa taattggcag atgaagcaca agttgcaatg 46200 tttctttgcc attcctgaggctccatcaaa aaacaatttg aaggggctga tcctaaacat 46260 tagataggat ctagaagactatctgtcatt aatgctcgaa gttcatcacg tagcatcatc 46320 tgttctaaac atagtccaacaatagcaggc ttccttcctt aggtgggtct tgtacgtaaa 46380 ggaaaggttg ggactttggctcaccctggc ttaagagggt agaaatagtt tggtgggggt 46440 tggggtggag acgagatgaccataataaaa tttttttaaa aacatgtaca acaaccctgt 46500 tcggcacctt caaactaaataagatataag tgatatgctt tgttgattgt caacgattac 46560 atatacttct actctagacctatggatgga acaatctctg ctagaactgg aaatgcattt 46620 tcacatattc tatataccagatggctgatt tgaggaatcc actctgtgga tagtagtatt 46680 agaatgtggt acccaaaatgtagttctttt tgtgcttgtg aatttgaatg ccccaagagc 46740 atcatagcaa attctaagacttacagaaag gatccagtag aggaaaaaga acaatttctg 46800 tacctcattt attttggagtccaccaatgg ggcagttttg tatttttata atggccactt 46860 tcagtttttt ctcctagacataaagtggag aatttacatt agagcttttc ataagcaggg 46920 atcacctctg actcctttgtgcccaatgaa caagtgagca taaagagaat cagagaattc 46980 agatgtaggc attgtctagttgtcccttca tttcataggt aagggtgcag aatgccagat 47040 cgagacagtg acttgctcaagggcacattc atcttgcaga acaactggga ccagtatgca 47100 ggttgtctga cttccaccagtcaggcagaa aagcagggtg ggcaatctgt ctcttgtaaa 47160 tgcaaaggaa cttgtgagagaaaatatggt agggcttgga aaaattccct ttttgaaaat 47220 aaccactgac tccttcatctcaggttgtgg cttcctgggt gagggcctgg actattgtgt 47280 gtgtgtggta taattactgggtaggtttat ttttttatgg gagtacaata aagagaacat 47340 ttcctgcctt actctgccatttgaaaaaga ttttcatttc cccttttggc ctggggatta 47400 caaaagacag actcctttccaattacagct cggtctgagc ttgaacatat aactcagcca 47460 gcagcaagtt caaattgattttttttcaag gctgagaaaa cgaaattggg tcttcttgac 47520 acaaaaactg gcttggtttttttctgtaat tgtgaggctg ttttcaaagc tcccttctaa 47580 agcagaagat ataatcatccaggactaacc acagtcttca ctgtaagttc tataggggct 47640 cagaagaaat tagttgggtgggcccacatg acaggggcac ttggctggga gaaacatttc 47700 cctgttgcac agtggcagagaaggactttc tccagacctg tgacctttaa tgtgctggct 47760 gggggtgagg agagggaggaaggaggaatg ggcccagagg gcagagtgac cttgaagatg 47820 gcagatgcac tgagtgctttctgccaggca ctgcgccccc attttacttg tgttatctcc 47880 cctaatcctc ccaatcaccctaggcgggag aagaaggaac atagtgagaa aagacagcta 47940 cacagcttcc cttctatccttcccctggga gctcaggagc tgtcgtagga gtgtggcttt 48000 ctgtctttac cctgagtaaatcctccacga ttaaaaaaaa aaaaaaaaaa aaaaaccttg 48060 atgtgaatgt gaatccataccattcaataa caaggcagga tttttcttcc tctgtcttca 48120 gcagtttact cttttaagggaataaaatga aagataagca agggttagca ggaacatctc 48180 tcgaagatga ctttccaaaggattcagttc tgtttttcct gtcttccctg attatgtcta 48240 aattacatac gttcacgattttgactgaga tttcagccat ctccattcaa tagcaaactc 48300 catccctacc gatcgtctatgtggccttgg aaaagtcatc tgaagtcttt ggatctcaac 48360 tgccctagtc tgaaggagttggatagagca ggagggggtg ggggcgggga catggggtgg 48420 ggacagcctt ccaaaaagggtgcttcttca actgcttttt aaggtaaggt gctgagaata 48480 ctatggaaag atgctacagagaagttgtga gaaggcagag cccagaagca gctagcaaag 48540 gtggtcacag gtggttcttcctttgtggcc tgattttcct tgcaatctac agtgctggcc 48600 cctcccctgc agatccagctgcctgtgttt tcagaataga atgctagtcc acaagctctt 48660 tcatcctctc ggcaaacatttatccagccc gtgctgtgag ccatgcatgg tgctgggaga 48720 gaggactgta gtagcttgtggagtctctac tcagccattg gaatttttag gtgcaaataa 48780 aagatttagt tattttcaccttctcctagc cacattttag gaacagagtg tctttcaagc 48840 ccatcttttc taatttattcactttaaaaa taacaagatt aagacccaga gagggaaaat 48900 tacttgcccc aggtcacacaactctttgtg ccaaaactag gtctggaacg cagaactctt 48960 aatcttctgc tttttattacattgccccaa attgcttaaa gggctaccca gggaattcag 49020 ttcaccaggg aacatcttgcttctccaaac attcttgcta acaaagatct tactgcacaa 49080 gtataggaag atgtcactattacaaattca tagcactcat attaacggca aagctaaaac 49140 attcctatag tcacccgcctccaattaatt aaaaaggaat gtgttgtttt tgaagtcttg 49200 cccttccaac cagccttgaagtaattttaa atagaaaatt ttaaatggaa attaaggcag 49260 ttttgtgata agagtgcctgccctttggtc tggggcatcc attgtgtgag ctagtttttc 49320 agagctgcag aagtaacaagttggcaggat ctcacatagg agataataga taaaaaccta 49380 catgtctggt ttcaggacccccaggaagct attgttgccn cttcttcctt ctttcccagt 49440 tctttaatca gtttctggcagacacctgca accccactat tgaagcttca tctcgttgct 49500 tacatctggg ccatgataaataaaagtacc ccaggaggaa taagtgctta gcagttacca 49560 acaattcata cttaaatgtgaatgctttca gagttggcat tgagagatac aagtggggag 49620 aggcatgctg gccaagagtggctgaggggt ttagagatgc atgatggtgg agatagcaga 49680 ttgggaagac acagagacccccagaagatg agcatttgac tgccttatgg ctttgcctgt 49740 gactaggctg gggtcctcagaatggaggaa gaaagggaac tagagaaatt ctgcatgggt 49800 tttcctaata gagcatgaagaagatctgct tggaattccc tgcacaccca ctgatatggt 49860 ttgaatgtgt gtccccgccaaatctcatgt tgaaatgtga ttcccaatat tggaggcagg 49920 gcctggtggg aggtgtttggatcatggggt tggttccctc ctgactttgt gccctcccca 49980 tggtaatgag tgagttctcgctctgatagt tcacaggaga gctgtggttt aaaggatcct 50040 ggttcctctc tctctctctctcttgcccct gcttttgcca tggaaagtgc ctgctcccat 50100 tttgctttcc actatgatcataagcttcct gaggccctaa ccagaagcta agcaaatgct 50160 ggcatggcac ttgtacagcctgcaggacca tcagccaact aaatctcttt tcaatataaa 50220 ttacccagtc tcaggtatttctttacagaa cacaaaaaca gcctaataca cctgcattgt 50280 gcaatccagc ctccaggcatgtgcttatgc catcacttct gcctggatgg ccttacctgc 50340 cactttgtat gaataaactctcttaccttt gaggctcaca ttaggcattg ttgctgcccc 50400 aaatccttcc gtgaacccttaggtgggctc cttcctgtcc ccactccacc cattctctac 50460 tactactgca cttcagtgatgacctggcca tcatgtcttt accttttgct gcctctgttg 50520 tgtgatagct tctcaagagagggaccatgt ctctttcatc accacatcac tacatcccca 50580 gtctctggaa ccaagctggcccttggtaaa gatgcccatc tggctgtgtg gatccaccct 50640 gcccaagtgg ccaagtgcctgcatatagtt gagagtggag atacagcttg ctggtactcc 50700 acgcagccaa gaaagcccaaaacaggacct tatgaggacc ctgcattttt taagaagcca 50760 catttttcac catggctgactagaggaagg tggctggaga gaacaaatgt ggttgggtac 50820 atatggcctg tggttgaccacatgagaact tgagcccagt gactagggtt tctgtacccg 50880 gtcacagcac actctgtcaccctcagggaa gacccacctg gcagaactta agagttctgt 50940 gtggagatgc aaccataaaaatatcagttg gcacgtgaag ttcttggcat tgtttatggc 51000 tgcattgact tgaggttcttcagacatctt gatccaaaat gggctctcca tgccctgaac 51060 aactcaaaat ggtgcacttctttcagggcc tgtgcaggga tggaaggtga agaagaacag 51120 tgtagaaatg gggtgggaagtaggagtggg gccaggatgt gaaggagttt ccagtttagg 51180 taaggaaaaa ggaaacaaaaagaggaggga gaaacaacgt cctaagtgct actgtcggtg 51240 gttggtccag cccgtcacacatctcctgag cccagagact ctgatgctgc aagtctacag 51300 tgaggtcagg gagctcatttgtttctttgt ttgtttaaca agctcctttg gtgattctgt 51360 tggacatctc ccactgtgggcaccactagg tcagaagttg tcagtcctga ctgaacatta 51420 gaatcacctc gagaactttgagggaaaaga attctgatgt ctgagcccta cccccagact 51480 aattaaatca gaatatctgagagtggggcc tgggcattcc agtttttaaa atgcttagtt 51540 gtgtggaatt ttcgagctctaaaaacatac tttgttgaca tatcataaac tagtaatttt 51600 gcaaaaactt ttttcataagttctcagatg tacatgaccc taattaataa acctcaggac 51660 tcagatttca ttttagtgtcataagctttc aatttgttat atttgagttt gggattcatg 51720 agtgattatt gaagccaacagagagctagg atgacagatt ttgggatgtc tctaaagtct 51780 ttccagccca gcttatcctattaagaggct atattcaaaa ctcaacattc agacaaccag 51840 aacagaaggg cagatgagtaataattgagg aatctcagct atgtcaggat cccaaacacc 51900 aacctttgta ggagtagcagagctcaatat caagatatcc ttgtttggta tgaggattat 51960 aggaattgca ggcttattccttaaatatag ggggaatgga ttcacgatca agagctgcca 52020 ttaatttcca tagcatcttggataacaaat aagaaaaatg tcaagagagg aaagcaaagc 52080 ttttgacagt ggcctcattaagtggaaaac atatttcctg tattaggatg gggattctcc 52140 aagggaggct taagtgaggagacctcttct tcaaattata ccaatgtgga aaggagttct 52200 gtttatttga ggctatagatttgtattgct ctttggtttc ctgtgttttg gtacatttct 52260 gaggtggaag aattccagatttcaactttc ctgaaccaag cattgagagc cttaatcagg 52320 tctaaagaat aggatgacagtaccttaaag agctgtgaaa atgtttccgg atgttgagag 52380 aacacaaagg cttccaataaacttcaatag aaaacttcca ggaatctgtc ccagacctga 52440 tcccttagct tcaaccatctatattccttg ggttccagcc cttctcatct ttgactcttg 52500 aactacttga aaaaggtgtttggttggagg gtgttgcttt tcacagaaat tcaatagtct 52560 tatagagaac agacatgcatgtatctatca ttgtaccaat agttaaccaa acatgagtca 52620 cttttccctc tctcccttcatagtaaatgc atgactgacc tagtttagga acactctcct 52680 ctggccaatg aacaatagctgtagttcttc atgtattttc tttctttgtt gaatttattg 52740 cttcatacac aaaaccctgaacgctaggta ctgttgaact tttgtgccat caaagagagg 52800 tctattgact cttcatcaaggcataatatt tcataaatag ggtgtttaac aagctcttcc 52860 taaatgtttg ccttctgcggccataaagac agtagctgta aatgtataaa gtaaatacta 52920 tatgttttat acatattgtcatccaattat acaactctgc caccaatttg aagataaggg 52980 gcctgaagct aagtgtgtatgtgagttgcc tgctgagtaa caaaccagcc caaaacttag 53040 tggttttaaa caataacaacatttattttg ctcaaaaacc tgcatttggc caggacttgg 53100 ttagaatggc taattttacttcactagatg tcatttggga caattcaaaa gctgggggct 53160 ggaataatct gaaatctcattcattctctt gtctggttgt tgaggcaatc tgtcatctga 53220 aaacgttgct gagactattagctggaacac tacagagaac ctcttcatgt gctctgggct 53280 tcctcacaac atggtggctaggttccaggg gcaagcatcc caaaagagac agagagagag 53340 acacaggcag aagctgtatcaccccctatt acctaatctc agaagtcaca cattgtgtca 53400 cttctgccat attccattcatccaggcagt cacaaaagcc tgccctattt taagaagaag 53460 ggaaatgtgg actccacctctagctggggc tgtggaaagg ttatgaagga catacgggat 53520 tggaaatagt gctgcagacattttttctgc tggttggtgt caggggagga atttgaaccc 53580 aggtttgcat gagtctatacaatgccctta atactgtacc acagtattct gtgaagctga 53640 ggactgaaat ctgcctctactgcaatggaa tttagctgtt aaatattaca cctgggctag 53700 tgagcagaaa cagccaaacttggaggcaga gagtgtggac tttgggctct cccaaggcat 53760 ggtgaaggtt gataatattaacatatgaga accattcttc cttcccaaga ttggtggaat 53820 cacttggaag acataccaaggccatcagtc ttacatttct tgagtactta caggtttctg 53880 agagtagata aataaaacaaatcactgagt cacattcatg ctaatggagc atagcaacta 53940 gattattgga aggtcagatcatttagtttt gaaaaatagt aataacaagg catattgtct 54000 gacttataag aaaacctcaagtatctagta actggcaaga tatttaattg aaatgaggca 54060 aattctagaa taacatatgacattgcagtg agataggcta ggaggtggaa ccaacaaggg 54120 agacttgagc caagcaagcctttcaacatt ttcccttgtt ttctttcctt ctctgacttt 54180 cctttctact ttttttttttaagcttaagg taagtagcat gtttataggc ctagaagcca 54240 gagttattgc tgacttgggaagnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55020 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55080 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55140 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55200 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55260 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55320 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55380 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55440 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55500 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55560 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55800 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56100 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56340 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56460 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56520 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56580 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56640 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56700 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56760 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 57960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58020 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58080 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58140 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58200 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58260 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58320 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58380 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58440 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58500 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58560 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58800 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 58980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59100 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59340 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59460 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59520 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59580 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59640 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59700 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59760 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60420 nnnnnnnnnn nnnnnnnnnnnnnntgagcc ccacctggtt cttctcctgt tgatgtttca 60480 gatggtcgtc acagatcacgatatttaacc catgagttta acaaacaatt tcttagggcc 60540 gcaaggctgt tgggatttgaaggggaatag aagaagctga aagagggaca cccagacagt 60600 gaaagtaaat ggtagcatcctcgcatattt ctattggtct ttgacaattt ttgtttacta 60660 aacccagaat catagtttaatacaagatta ataagacata atgaaaatgt agggagccat 60720 gaaggatacc atgccctatgctcctggcat agctgattgc acacggatca gaggatgtcc 60780 atacaattca acaccagccctccgcagcac ctggggagga cctgttacct ggctggaccc 60840 tcaggctggc tccaggcatgcagctcagag cactggatct ctagccagac cacctgcgtt 60900 cgtcctggtt tcaccacgatgataacctta ggccagttat tttatctctc tgtgcctcca 60960 tttccacatc tgtaaaagctgaaaatatcc cacttcacag gcttctcatg agaaataaac 61020 aagttaataa catatgtaaatcctttagaa caatgcttga caatatagat atgttattat 61080 taacattatt attatatgaatttttcatgt agttgagaag tccactattt tgctagttcc 61140 tccttcaatt cccttatcctcccaccacca ccaccagagg aaaacagata acagtatatg 61200 gatctggctt ctgtttttgtttttgctttt ttcttcagtt ctcctcttgt tacttccacc 61260 ataactggcc ctggggaaattcttcctgtg tcgggatgtc ttaaaggacc gagaacctgg 61320 tacctgtcaa gtgcttcttgagaagttcct agggaacgta catcataaac tcaagttagc 61380 accaagctat tattaaattgatagccacct gtctgagcat gctttattgg taactttacc 61440 tccgtgggaa ggtgtagcgcccacactgta ccctcctacc tttcccattt tatataacgg 61500 agtccgtgct ttctcaagactcaggggtgg gtctgcttcc agcactgagg aaaagtgaga 61560 actgaatagg gagtggggcttttggtgtag ttaagaggga ggtaaaataa ctgtaatttc 61620 atagcaatta catacagccaacactgtcaa aagttccagt tttccattat aaacctgagc 61680 ccctgctggg agcaatttgcaaggtagaaa aaagcacaca atgaggttct aatgtatgaa 61740 agaaggcatc gctgcctccccatctgaatg ccatagcaaa acaaacctca agggagaaaa 61800 cagtgtgttg agcttcgagaacgcatttgt tttctccctt tcttttgaaa aaaaaaaaaa 61860 aaaaactcag taagcccccattcctggata ttggtcggat tgtttacagc tttgtacgag 61920 tcttcagtac cctggcccacacaggccctt cctgtcagtg gaatgccctg ccaggccacc 61980 tgcaggggcc atggtttccagggcagccaa catcagagtc tggagcaaat gggttatttc 62040 ttcctctaag tattatgttgggggccaata aaccacccta tggaaatgaa ttctacagag 62100 accaattccc caaccttttttgtttgttca taggtttgct tgaaattaat tctttatgaa 62160 attcagggat ctgcagcccactcagaagtg cagtaaagat ggacttgctt ttcagaccta 62220 aaaaagccta ctgttttgaggaagggaaat gaaatgacag aagcacagaa cacccaataa 62280 taattctgtt atttttacatatgttcctgt cttatcccca agcccaccta cagaaggcca 62340 taagctcctt ggaggaattcaattttcaat cccctgggta attttctcca gtgtcttgca 62400 cataactgat agtcaatatgtatttgttga gtaaaagtgt aacaaatgaa tggattattg 62460 acagtgaagg ccaagtactagccagaacta agtggtgtat tgattaaagc cttgagcttc 62520 ctgattagcc tccatatagggagatgggat caaagtctga gatatttata ttcatgacat 62580 ccaaggactg aggggtgggggagggtaaac tacagaggaa catattttgg ctaggtagcc 62640 gaaggaaagc atttctaattgttagagcaa tcctcagaat gaactgcttt gagagatatt 62700 aatacaactg actggtcagtggagatattc aagcagaaac tagatgatta cttgggggtg 62760 tagatgttgg cattccaatatcatttgtgt cagactaagt gatgttgcag gtctattttt 62820 accccaaggg ttctatgatttcataaagtt atgattctgc ataatcatca agttcaattc 62880 agcaagtatt tattgagcccttacatgctg ccaggcattg ccctgcgtgg acattaaggc 62940 aaaaacgaat aatatcaggctaccgccctt tgaagttgct gggacatggt tttagtaagg 63000 tctcctcaga cctgcatttgggtgttacaa tcttatgttc cccttgctaa ttgcacctgc 63060 ccactcagga cactggttctcttctttcca ggctggaatc gatggagaaa gcatcggcaa 63120 ctgtcctttc tctcagcgcctcttcatgat cctctggctg aaaggagtcg tgttcaatgt 63180 caccactgtg gatctgaaaaggtaacgtac gttgcataaa tgaagagtat gtttagctgc 63240 atgaacagag aaccttccatgaaggagggc aaaagccttc cgtggctccc agcagccccc 63300 tgctcacacg tcannnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63720 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63780 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 63960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64020 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64080 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64140 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64200 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64260 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64320 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64380 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64440 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64500 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64560 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn naaattagag gggctaaacg 64800 ccatctgctt ggttcaaatactatgctctt cataatacct cagttatgaa gtttgtttca 64860 tctttcaaat gagaccttcaatccctcaag gacagactgg aatcgtgttt taagtttgtt 64920 tgtatcctcc atagaaactagtttttcaca ggtcctcaaa tactatctgt caaataaatg 64980 aacttgcatc cactatgcaattaatgtctg tgataactaa agagagcagt agacttatga 65040 aaaagcagct tccttcagtgcagaattgga ggtgtttgga ttaccatgtg tgagagagga 65100 cttgtatgtt tgctggagaagagagtggga gagggaacca cccctgcccg cgtgcttcag 65160 atggtttggt tcctccttcacatgatcaca cctggtccag ccaacacccc tttgacgtag 65220 accaggcatg tagcaccactccatttttac agataaggaa actttcttac tgccccctct 65280 acccatgaac acacacattaatttctaaga aaactgattt attttcctgg cactacactt 65340 tagtgactga ataatcttgaacttttcaga gcctcagttt cctcatctgt acaatggaga 65400 cagtgtcttc ttaatcaataggattattgt gagaatttat cgaggcaggc catgtaaaac 65460 atttgcaatt ggtcctgtacctgactcata gcaagaactc gttgcatgtt ggttattctt 65520 agaattatca ctgggttcatggagctggca atccagtaag gaaaagaaga ccgatcctca 65580 ttacataatt cataaatcatacctggcaat acgtcatcca atgctaatga tatagtctca 65640 actataaata ctgaagcagggtcaacaaag acagagggct gcaactctag ctaagcgtcg 65700 gtggccatag gcagtacttcccaagggcgt ctgactccag tggccacaat tctccagaat 65760 cccaggaaaa aacccaccaccaccaccatc accaccacca cacattcctg tatctgtaag 65820 ccacagagag gtgactgagagcacacacgt tgctgccttg tgtcagctct aaaaaacagg 65880 agcaaataaa gaatgttctttcattgtcta aaatgaggga catgcacaaa gtaaacaaag 65940 ggactaagta aattagattttaaaaatact ttccgtctca gttgtctgag ttccaagtgc 66000 ctcagaatgc agctggagagtgtgtgtgtt tggggaggga catgggagta gcacagtaat 66060 tgaaattctt ctcttcccctcccaaactga ttaccccagg aatttgtttc attacagact 66120 cctctactcc cactgtttactttttttaca gtttctgcgt ttaatgtttt ccactcccga 66180 ctcccacaag aaatgtggcattctcagaga gagcatttgg ggtgaatccc agcaccgtct 66240 tggaagtcgc caccattttccctcctcttc cctaaacatt tttagtgtaa ccctcccctt 66300 tcccattttc cccaatctgccaaaccttct tccccctcat tttgctccat tcttgctctt 66360 gcattctgac cagctggcgcagacacagaa gcacactcct ggatattaac aggacctagc 66420 cgtgtcaaac acaaggccactttcctttcc actgcacttg gccttcctgc aggtagtcag 66480 tgtgctcccc agtgcacgccagaaatgcca cttagcactg ctgtgggcta agtgcaggaa 66540 gttcaattaa ttgttcagttaattaattgt tcattcattc aattgggact gcgttccaga 66600 atttcaccca cttctccatcatctaggcct ttctttcagg ataatttagg actcttgggt 66660 ctcctctttc ccaggggggagaaatgcact gttgctttgg ttatcttgga tttctttaag 66720 tgtctgaccc agaaactaacaggcacacca aagagctttc tgtcttctta gggagcaaat 66780 ttgctgccta ttacatatatatgtatgtgt tacctaaaag atcctactgg gcccagcaaa 66840 gtttgctata agaagaaataactttcataa atcaagtgat ctatttgaat cccatcacaa 66900 agcagaagtt gcatttacttagaaaataaa ttgtttttaa cttaagataa catttaagta 66960 accccataat ttttaaaaataggcacaatt tctgactcct aggagatttt tgatttatgt 67020 atgttgaaca gaatttttaaaatttaaaga aaaataactt ctttctagta aatctagtaa 67080 atgttctcaa aataagtcttattttgagaa cagtagatgc ttgaattaag ttaaaatact 67140 gtgaaaggta gactatggtggagcatgaat actgtagcag tggagttgcc tactggttct 67200 ccccacattt aactgaaagccgaaagcctt cataactgga ttgaagtttt taccaatttg 67260 agaccaacat ttgatttctacttttgcagc taaaatcact catttttaaa tttaaaatgt 67320 atgagttttt attgctgttgttgttttgag accgagtctc actctgtccc ccagactgga 67380 gggcagtggt gccatcaacggctcacaact gcctcgaact cctgggctca agcaatcttc 67440 ctgcttcagt cttccaagtagctgggactg caagcacatg cctccatgcc cagcaaattt 67500 ttgtttgttt tttttttttgtaaagatggg ggtcttgcta tgttgcccag gctggtctcg 67560 aactcctggg ctcaagtgatcctcccacct caccctccca aagtggtggg attacaggca 67620 tgagccactg tagctgaatcaaaaatgcat gctttattca tgtagccatt aaaaatatgt 67680 atctactgag ggcctcctatatgccaggca tggttctggg tcctttccct catggagctt 67740 gaatcgtaag gctttctctcaggtatcttc ttactcacca gtaggaggtt cttggttcag 67800 aatccttggt aatagcctacctggtgtttg tggaggaata atcttagcaa taagaagact 67860 tttgaaaaca tcttcatttaatagttttgt tttcaatcta atagtttgtt tattttcctg 67920 gctacccaaa gaccatatcaggggctgttt ataactcaaa aagagtattc tgttgagtgt 67980 gacaaaaggc tgaccaatgttagacattag ttattagata ttaaaatata agttacacac 68040 aatagaagca tctaacttgaacaaatggcc caaactccca attttttgta tctggagata 68100 tttattagga gaaaagtatgatctaattca ttaattttta aagtatggaa ttatgtgtag 68160 cgcatttttg ttaggcagattggtgaaaga gcttttaaaa gcaaaacccc acctctcccc 68220 tgggtaatta aagcttggatctgacttaga tagtcacctg gattctctcc attttttcaa 68280 ttctttcatt ttttcaactccttcatcttg acttgttcct ctctcactga ctccactgga 68340 cacacttcct gggtcacaccacattctttc agaactttct ccaacctccc ctgccctata 68400 ctgcatacca taaatctgtttcacctgaca ggcatgttgg atagcacagg gcatctaagt 68460 tacaatcaat cttaggtttcccagaacagg aagcaccctg ctgtaagcca acacctgaat 68520 cttgctctct ctacagaaagccagctgacc tgcacaacct agcccccggc acgcacccgc 68580 ccttcctgac cttcaacggggacgtgaaga cagacgtcaa taagatcgag gagttcctgg 68640 aggagacctt gacccctgaaaagtaaggat ctgtttcttt ctggggctgt tgaatgggaa 68700 gggttcatgc tggatctataaagtcctggt gttaacttcg ggcagatgca tttccaaagg 68760 aacctcacca gtccagagaacaggaccctt tcttcaaaga gagagcctag gacccccaaa 68820 gggctgccaa tctcaaaaccactccacaaa atccactcaa gagtagatat ggcctatgaa 68880 caagagccag tcactcctacctacacagag tgattttagg agccctagga taccaggcct 68940 ccctcgcccc agtatgttccttctctccag ctcactttgc ttggaagacg ttgaagtgag 69000 gacaaagttt ccttcatttcagaaccagag ccgaaagcct gacagaatga aaataagtca 69060 ggaaagggta acccagtgacttaaaacaac atccagggtc ttaaacctga ccaaatctgg 69120 tctgagtcca caattaaacagatgaaaaaa atcaactaaa agcagcagct cctcttctgc 69180 aaacaggttc agggagagtatagaccatag agctataagt tcttgttggg tgacacattg 69240 ctactgatgg aaaagagcagtaaaaagaag acagatggct tctaaaattt acccaagcta 69300 gacttgttca ttaaagaatactattatcaa ggattaaaac aatcacaatt tttgaaaaaa 69360 ctagaaaaat aatattacatatagagtgag gcatgaactt tatcctgtag ggtattatcc 69420 tgtagtgagt cctgtgggatattctgaaag ggattaagca caggaggtgg ggagtccgtt 69480 ttaagaccta ctttagaggttgagggaagt cggtatccta gaggaccatg aagctggaga 69540 tagaggaatt aattctggggagccattggt gactgtccag gcaggacgaa tgaagggcta 69600 agccaagaca gggcaggaaaatcgggggag aggagtagga agggttcagg acatactgag 69660 taagtaaagt tagtgagatttgactactaa aaaccaccat cctgcattta ttttaaagtt 69720 ttatttattc atcaacacttttatagcctt cattttgtgt caggcaccat tcacaacact 69780 tagctaatat gattcatttcatccttctag catcctaggt actatcatta tcctcatttc 69840 acagtcagga cagggagagagtaagtcact tacccaaggt tgcacatcta gtgaattgct 69900 gttaaaaaat taaaactgccagtcatgctc caaataagcc ttaaacaatc atatattctt 69960 ccttaaaaat attctttgtcatccatattc agtcagctta tttgcacact gagagtgaaa 70020 aagccacctc aagcccattggaagcagctg agtatagact ctaaataagt gcattaatta 70080 aatcaatagg acagccccacatacacacac acatctctga agaggacacc agccagccac 70140 ttcactggaa gtgccctgagggagcccaga ctccatcccc ctccccggga ctcaacctca 70200 ttgctagcac tgatttgaaatcccacatga aaaaaaatgc tcttttcaat aactagagtt 70260 tagactttgc ctgggagacccatagaggaa agttcatgac gcagttaaca ttttccctct 70320 tagaagtaac actggcactcaggagtgggg gcttccgtca tgatcattag gaagtggaaa 70380 gtggaagcag tgaggagcagaggggcccat ttggtctgtt gccaggcagt ggatggaggg 70440 gtgaggggct ggggtacaatcctagtctgc tagtgatgtg ctgtgacctc ttgagccagt 70500 cccttcacct cttgaaccagtctcttctct gctctaggac tcagttccct tctctgtgac 70560 aggatatctc ccagagcccttcttgctctc tgattcttca catgcccttt agccaggttc 70620 cctcaccact accacaccacccccagtgca actgtgcctt tcctgggctg cttctctaac 70680 tcaggggtct ccaatctgcaaccacggtgg gaccacaagg tgggcagaga tcactggaat 70740 tgtatgcccc atgcgtccttgaacattctt ccagaggcga gtcatcatct gacttcgtca 70800 tcatttcatc agattctaagtttccatgat ccaaatagag ctgggcaggg ctggatgtgg 70860 accctccctc cactccctcctctccaccta ccttccctgt cctgagctct gctcccgctg 70920 gtccaggtcc ttctctggactgcagttgtg tataggaggt tttatgaagt gcattcctgc 70980 catgttagtg acctaacaatccactactct caggtagcag aacgcttgat gctgttaaaa 71040 tgcatatctc cagaagcctaccaaggtggg ggcgccaagg gtgaggccat caagtattcc 71100 agagcatccc aggattataccttagatgcc tttgcaccca tcagactgag gcaggtgcca 71160 caattacctc ccagtaaatgtcaccaagcc agtgcctcag ccccatagag acctgctggt 71220 ctgcccagaa caaaccaccagcctcaccag cttccccagc gactcaggct gggagatgga 71280 gtggctggga ggttgtcaggcctctggagc aggacagcct ggccttctgg ttctctaggg 71340 ggcagcaaaa ccctcataaccattcaccaa gcgctggtcg gccatgtgtt tctcatcata 71400 ccactatttg taaaagctgccaaaagccaa cacattatgg gaggcttttt cccttagctt 71460 tattttttaa aggagaaaaccacatacaaa cactccaacc atttttttca aatcacattt 71520 gatcccaggg agcatttggtgtcaaaatga ggaatccagg aagtatcaag ttgtttctaa 71580 atgagactaa catccatccctctccttaga ttgatctagt gaacacacga caaggaagtg 71640 ttgtgattaa tgctgggattactgttgaga cattctggcc cttgcctttc atctcctgaa 71700 gtggccaaag tttctaggattcagcactga gagtcaggag ccctgcctct ggccacctct 71760 ctatgagtgt ttgtcatatgaccttggaca agcctctctc ccgctcagac cctccttgcc 71820 ttctcatctc taaaatgagggggtgaaact acatagtctc caagatgtct tttgttgcat 71880 gttccacaat tccatgagtttctcaaagta ctcttccaga gctggttatt gtctcaaata 71940 atcctagcac atagatgctgtaataaaaat tcaggagggt aaactgatac agtctattta 72000 cacagcatgt catttagtcagtcatctagc aagaatgatt taagtatcaa cagggttcag 72060 aacactgaca gaattggaagacaacttgaa ttcattattt tacaaaatgt atagcgataa 72120 ctcaccacag taaaaaacaagaaagttctg accctctggg aggtagagaa acagcctcca 72180 gacctcccac aggatggcatggtagctggg ggcaggaggt ttggagtcag agtgactcgg 72240 gttcaaatcc ataccactaggttgtgctat gttgcagtaa caaagcggcc ataaaattcc 72300 agttgcttaa cagaacacaggtttatttct cacttgagca aattctgaga gggtagggtg 72360 gccctcttcc atcctgtagtttttggaata catgccctcc aaggctcctt cagggagaga 72420 gagaagaaag atctcccaaagtgttttgca gggccaggcc tgcaaaaaaa atgtcatggt 72480 gacattactt tccttcctatgccattggtc agaatccaca cgtgatccca acttaggtga 72540 gagagaagct gggaaacagaattacatggc tatttagtga gtatgcatat tctccgctac 72600 aggcaaatga cctgaattctctagaatctt taattctcac atctttaaaa caagaaccat 72660 actgttgatc ttatagggctattgtaaaaa ataatactaa tgagcctaga gcctgatatg 72720 aagtaagagc tcattaaatggaagctcaag gagttagaaa gagtggccag tgcctgtttt 72780 gggtctgtct cccaggtgcaattgtacaga caattggtga ccttaggtct taaatcccca 72840 gatcctaggg gatatcttttaatgaataag ccaggcctct tccacctcca gagatctttt 72900 gcttacacag cagacatgagtcaaacaagc acacagtcaa ggattagaaa agtcagatgc 72960 attaacgtcc ttcacgtgtgaggggttgat gaagtaagga agtgggaaca atggagaagg 73020 gagagagaga ggaagtctttccacttttca gctcgtgaga aaaaggaggg aaaacactta 73080 agctgccatc agcaaggccattccatcagg accgtctttc tttttctcca gggtatctat 73140 ccccaaaacc aatctcagggcctagtcctc agtaggtgtt tcatgcacat tggtttaatt 73200 gcatggtggg tatccttttgttcagtgtct tggttggctg tggctagtca gtgagaagtc 73260 agctgggact ctaaaccagatagggcactt accatctgca atggttttct ctttttttgc 73320 tactattttc tacttttgaaaaattttttt tttttttttt ttttttttgg tgagaagcca 73380 actgttttac ttcccagaatgaaagttaag tgcatgcatt tgaaataaga tatcctaaaa 73440 accgggtgga gagaaattttcatcagttag atcaggagtt agcagcttta tctgtaaaag 73500 gtcagatagt catgtatttttggctttgtg ggcccttcag tgtctgtcac aattccacag 73560 tgacactgtg aaagaagccataaactagta tgtaaatgaa tgggcattgc tgtttcaata 73620 atattttact tatgaacactgaaatttgaa tttcatatta ttttcacgta tcacaaaaca 73680 gtatcctcct tttgatttttttcaagcatt taaaaatgtg aaaaccattg ttagcttgag 73740 aacctcagac aaataggggtcatgggtcac agtttgctga ctcctgagtt agaaaaaatt 73800 atcatctctt tgattatcatcagatcacat ccaatgttat ttcaaagctg tttctgtgag 73860 tgaaacactc ttccttagccttttagagaa acagaatgta ctttgtagcc cgcttatttg 73920 catatttaaa acacgccataaatatggagg taaaatgaat gcacttatcc tatatagtat 73980 cctcccccag tgtagttactgaaagttgcg agattaacat cttagagacg cacttcccac 74040 aagaacaaag ctcaaatggaacactgcact acacagtaaa atcacaacga gtcctagtgc 74100 ttgggaaaca ggatgacaccaccacagccc cctgaataca cgatcctctg tcatcatctt 74160 ggatttggta ccacacatccctgtttggcc caacctgatt gtgaagaaat gatcagatga 74220 ctgaaagtct gaccaattgatgagatacag caaaacaaag caagaaaccc aaacaagctg 74280 tccactgagg tgcattttctaggcctttct gtctttacat tatcaaggag aaacaaagaa 74340 cacttggcat ttgttaatagcaatttaagt ctatcgattc taatagcatg ctggattcta 74400 tgtgagaagg ccaaaatagaaagagatgac actgaattcc tgtgtcttgg tagattacct 74460 agtatcttgc aggaagccctgctgtgtgcc cccagccaca gcagatgaat gtggtgattc 74520 tcagtatacc caggaagacatgtgtgtgga gcatcggtgc aaagcacccc ctcctgatgg 74580 atggagacag ctcctccaaggtgggcagct cagcctttaa ggttctgggg ctttgagaat 74640 caacagcgga gtttatgagaagccacacaa tgtggaattt aaaacctagt cttccacagt 74700 gaaactgcct gggttgggatcctggctctg ccaggcagcc atgtgacctc aggcaaattg 74760 cttaacctct ctatgccaccgtttcctcat gtaggaaatt aggctaataa tagtaccaat 74820 agagttgttc tgaggagtaaatggtgaata cgtgtaaagg gaatagaaca gtgcctgcct 74880 gctgcacagt aggtgctcaatgcaaattac ctttcactgt ttccagagaa gcagtggagg 74940 gggaaggaca ctgctgtagggccaggaaac caggttctca tcgtccctcc accatttatt 75000 agccatcaac atttatcttctcaggtatcc agttcccttc catgtaaaag gaggacaata 75060 atgccttcct tatctgcctcacagcagaga gctggactct tccaatggca gtatctctga 75120 aattacacag ccacctaacatggccccaaa aggggcccca aaaggaatcc tagctcatgg 75180 gccagttgag taacaaaaaagcattaaaga acaacaaata ttttttaaag tcttgtttgg 75240 ttcgaactat caaaaacatcaaaacaaaca aaaaggattt tgctttttaa acgtaaggca 75300 acccaccagc ccacagtcaaaaacgattca cccagagatc acagaggtgc acattcattg 75360 aaaataatga tagcctggcgactgtctccc atctcttccc accactacct ggccataccc 75420 ataaaatgct tatgaaataatatttatttg agttgatttt tttcattttg atggggctaa 75480 tgagtccaag ccctgggttcccacatgagc aagtttacct ccttcagtct caaggccaat 75540 cgttctaact ccagcctgcctcttgtatta ttgtggcttc aaaatgaaag actctacgaa 75600 agagagagag aggggggctggatcattgca aatgtatctc caaataaaat aaaataaaac 75660 aattgaaaac acatgtccttttttggagct tggtagtttc tgcatgttat tactgcacac 75720 aaataataag catgtaagggcatttcagaa gaggaccgtg ggtattcttc ctttggagaa 75780 atggtttaat gctaacatacctggcttctc cttgctccca gaattagctc tgtgtagcta 75840 ggattataat cacaggactggcagtagatg gttattttag cgggtaaggg aagtgttgag 75900 aaccctacca gatagagaggcaagctcaga gggaactttg attgcgccat cccactggca 75960 catccttatt ggtggtcccaagaaaaggac acatcctgag acccctgaat ggcattctgg 76020 ttctttgcct agcagactgtgtcaccctag gcagatctca gcacctcact ggcctctggt 76080 ttcttctatg gatgccctagggctgagaat gcccaccctc cctatagtat gaggataaag 76140 acccccaaaa cattttgtagatgcagctgg aaagggaagt attgcttatc atgataggga 76200 taggctcacc cttaaaggagctccaagtct actgattcac agaacactgc atcctccatt 76260 ccaggtaccc caaactggctgcaaaacacc gggaatccaa cacagcgggc atcgacatct 76320 tttccaagtt ttctgcctacatcaaaaata ccaagcagca gaacaatgct ggtgagtgac 76380 tcccttccac caagaacctttggcagtggt ctatctgctg cccaaggcat cttcaatttg 76440 cccataggca tgtcaaatacttgggccaga gaagcatctg gaaccaatac tgcttattat 76500 ttgcccccac ccccccgccaccaccatctt gtcccccttg acacagggga ggaggtggtg 76560 gatgctgctg cctagctggtgcaggaggaa aggcttgggg ctttactgcc aagctggaga 76620 gagggatgct ctttgtcatctgagcctgcc ttattgcaca gcatagtgca ctgcacggaa 76680 cggtcctcac tgcccatttgtcccaagttc ccaaccctgc ctgtgatcac attagagcaa 76740 catattcccg cttccttctggtccctacaa ggtttctcca gggatttttc cagaggccaa 76800 gggagaggca gcacagtcactactgtggca aatgtggaag gcagacctgg gctgtgttct 76860 ccccttctgc aggacagtgggccctggaaa gccactcctc aaagcctggc ccatcctaca 76920 tcaggccctt catttctgacattggctaca gaattcagga cttggttctc ttgcggggcc 76980 ttctgaaagg gagctgagtcccccccagtt atgctcacag cactttcagt ccttcatttg 77040 gcattgccct ttaagaattacctaaagcca tttgaatgga accgtccaat taagcaggta 77100 taacacttga agaacggtgttaggcccagg aggaacaggc tagccactgc agtgccaacc 77160 tggggggctt ggatttttatcctatgggca atggcagcca ttcccttgcc ctcagaatgg 77220 gatgacagga cagcctcacaggctggatga ctggagtcca gcacaaagcc aagcaactta 77280 agtgctccat gaatctttgtgaatgaacaa gtgactgatg cacaatggac caaaacaagg 77340 gaatagatgc tagatgccccagaagctcca aggagaatag catctctttg gatcaaggcg 77400 accagcatat gcagctatgcatggtaaaag agacgaaatc atggcaggaa cagagttggt 77460 gtatcgggaa gtaaataattcaggaagcta ctcaggaccc agattgtgac aagtgcaaaa 77520 cttggttctg gaggtccaaagaagaaacaa acacttcctc agtcttcaat gcctggcaga 77580 tgcagaagga caggcttcaagtcagtttga tccaagaggg tctggagagc tgattaactg 77640 caggtaaaag aacaaaataatttctgtcga aggtattcag aaccagaagc atttaccatg 77700 ggggtaaatg catgagactccttttctggg agtttcaact gggtcataaa gattaagaag 77760 gaagcagttg gctgagatcattgagcagaa gatgcgggga agttgagaaa aagtcaggga 77820 cagagactgg aaaaagtgactgaagcccac agtgccagct tggagggctt gggtttttat 77880 cttgtgggca atgacatccattagaggtgt gtgtcaagaa actagaaaga gagtggcttc 77940 tgggaaataa gaagaagcatgtggaaagta ctttcaatct acttggtgct cttgcttcat 78000 tgctgcttga accctatagtgtgttgagta gtgtcccccc agaattcatg tctagcctgg 78060 gcaacatagt aagaccctgtctttacaaaa aaaaaaaaaa cttgttttta attagctgag 78120 caaggtgatg tatgcctgtagtcccagcta ttcaggaagc catgacaaga ggattgcttg 78180 aacctgggag gtcgaagctgcagtgagcta tgattatgcc actgtactcc aacctgggtg 78240 aaagagcaag gtcctgtctctaaaaaaatt tttttaaatg tatgtccacc tgggatctca 78300 gaacgtgacc tcatttggaaatagggtctt tgcagctgta attagttaag gatttggcga 78360 tgagatcatc ttgcatttagtgtagtccct aaatccaatg actgttgttc ttataagaat 78420 aggagagggg ccaggtgtggtggctcttga ctacatccca gcactgtagg aggccaaggt 78480 gggtggatca cctgaggtcaggagtctgag acccccgtct ccactaaaaa tacaaaaatt 78540 agccagatgt gatggcacatgcctgtagtc ccagcttctt gggaggctga ggcaggagaa 78600 tgacttgaac ccaggaggcgaaggttgcaa tgagccgaga tcacactgtt gcctgggcaa 78660 cagagcgaca ctccaactcaaaaaaaaaaa aaaaaaaaaa aagaggagag gagaggacac 78720 agacatgtag aggagagaaggccatgtgaa gacagtcaga gactggggtg atgctgtcac 78780 aagccaggga actcttagctttccggaagc tggaagaggc aaggaaggac acctactaat 78840 accttgatat tggaactgtaaggggaaaag tctctgttgt gttaagccac ccagcttgtg 78900 gtactttgtt gtaacagtcctaggaaatga atatggcccc tcagagccat tgtgggaaag 78960 aagagtcatt gctgctttcccggctgagga cagagaacca aacgagaaga cccatgactt 79020 gtctaccccc aaggcagagctggctctcga accctcgttt tcagactcca cacccagtat 79080 cctttcctct tagccacaaatatcttcttt catacttgtc aaagtgccct cagaaagatt 79140 atctcatttg agccctccagatttttaaaa ttataaatgg tgtttacata ttatccatat 79200 ttacagttct cctattcccaagtccaaagc cagtttccac caaatgccgc atatccattt 79260 cttataaact cagtactctccaattttcat tgtttcccct tcttctgttt caccctgttt 79320 tcttctgtaa tgagacttgcccttgtgact cctgcttccc ctaactcatt tgcacatttc 79380 aaagctccaa cattgctgctttcccaaaga gcctgtcccc agatgtgcta ttccaccttc 79440 tgtttaacaa gacaccacacattccttccc tggaactata acttgcccac acccctccct 79500 tcctctttac ttgtccttcctcccacccaa tacccccgag ttgtggtcaa ctgggtccaa 79560 aattcttagg cttagcaagccagaggtgaa ggctcctgta aggcttgccc tcctgtagag 79620 agcagcatgg ctcctgtgttacggtcccag agccccacag cccctgaccc gggagatggt 79680 ccatcccctt tctgctctgtgtgagctcca tggctgtggc ctctccccaa gagcatcagc 79740 attcaggccc aggcctccttccacatgccg ccctacatta ctttgccatt tttttccctc 79800 tggatcttca gctctgcacctagttctcca actttctttc tttagagggt ttctcttgta 79860 ttttcattta ctttctcttgagcagagccc tccctcccct ctacctggtc actaatgccc 79920 cagcccccat acacacactggcccaggtca tgatcctttt ctgccctctc aaaatgaaag 79980 catgagaaaa gccaacactgttacatggtt ctccatggtg ggtcacctct ccactccact 80040 cctgtggtgg ctacaggaaccaccaagtaa aatttccctg ttgttcccca aagccttgta 80100 agtccccagc taccacaaccgagaccccat gccgtgggag ccatcccagc agtactggag 80160 taatggagag aatcatccattagtttgtac attcagtcat tcactgattc attcaaaaaa 80220 ccacaagtta tataaagtgactacaagtcc gagctaatat ttaaagggca ctgcttatgt 80280 gccaggtact gtgctaggttctggagttgc caaggcaacg agacaaagtc attgccctcc 80340 atgaactaac aatctacagggagctgtaaa acaagtacat gtgcaattcc cggcctgggt 80400 ttgaatccca gcacttctagctgcacaact ttacccaaga ggcttattct ttctgagcct 80460 ctggctctgc atctgtaaaatggactttac attcagtttc tttatctggc aataaggtaa 80520 ttaataatat caccaaggtttgtaatgcag gttcatctat tttataactc agcgcatgta 80580 aaacccttag cgcagtgtctggcctcgtaa ctgttaaatt tgattattct gattattgct 80640 attagcacaa aaatgggactaagatgaata atgacatcag aaactgctgc ttcagctctt 80700 gcttccccct cagttagctcttccaaatct cccannnnnn nnnnnnnnnn nnnnnnnnnn 80760 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81060 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81120 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81180 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81240 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81360 nnnnnnnnnn nnnnnnnnnntagacaattg taccttagca gagagttctg gtataaaaat 81420 gcagtttgag gggttatacatctttagatg attaagacga tgcccaagga tcaatcacat 81480 agatgggatg acagagggagaggagtcaag gaggaagaga aagaaggact agccagggag 81540 gatggaggaa atcgggggtagtggggaggg cataaagatg ggaaagcaag ctttattgtt 81600 gttgttgttg ttttaaaagagtgatctgtg ttataaaatg ctgcagagaa ggtcctggga 81660 gacttttgcc tgctatgtgcagctccctga aagtgttctt ggatatcctt ttctgtctgc 81720 ttttctcatg tggaatccacccattactca gattctacac aaccagagag agctcagtct 81780 gatggaggaa gcacaaaccctgctccctta gaaaggggag gaccagtggg tgatgatagg 81840 gaactggatc aggaacccacatgggaagag tgctcgtggt cctgcgaaag agcatctttg 81900 agatgggaga aagcagcactgcatttagat ttggctggag gggttcctcc tctgagcccc 81960 acattcactt tttcaatcttgcagggagaa gttatgccct gggtttagac ctggaggttc 82020 atagagtgct gaagtagaaggcttgaggga gggcacagtg tcatccatgc ccctttggca 82080 catcctgcag catccctgtaattagcaagc agttaggtct gtcctccccc ttaatcatgc 82140 tctctaccca gcctgcctggcacatggtgg agttcagcaa gggcccaggc ctccaccttc 82200 tgcaagccag tgcctccacctctagcaagc gtcgctgtga aacacccctc cccgcctgcc 82260 actggaacac ccctttggctccttgggttt gtctcttccc taaaggggtt tctttgacac 82320 ctactttaac atgtcccccactctctgtca ttcctaagag gatgtggact tctccctctg 82380 aatatctgtc ttacattttgcatttcttac cctcaatctg ccatttgaac agactctaaa 82440 aatccctgca aatctcttggttttcaagaa attcatttta gttacttatc ataatctagg 82500 cagaaagtgc tatatttcactttattttcc acaatctctc tttccttatg cttcaatcct 82560 agcaatacac atgatttannnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82800 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 83040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 83100 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnncctcc ttttgcgcag 83160 cccacttcca caggaactccctttccccta gtgagaggtc tgaaaagccc caggggcaat 83220 agtatctcat ctgtatcacatgcagtcttg gggcttaagc tctctcaggc ctctcaggcg 83280 acctgcacta agtggtgttttgcccaatct ggatggtttt agtctttcaa ggcttccttt 83340 gaattccctc ttttcttcccccttcaagtc cacttttctc tcttatttcc acctctgtat 83400 tcctctgtct tggggggccttctttctatt ttaaaattta tactccactt gttccagaaa 83460 ggctttatag ctactcccagggatgcataa agcacggcaa accaacaaag gccaagagga 83520 ggtgagaaag aaaaacaaggagacaaccat caaatggagc cctgggtgag ctaaaacagt 83580 cagggcagcc ccggggccttcgtgcgccac acagaaggta gaccacaaat ttggctctaa 83640 acttgtgagc agacaaagcaaaacagggcc cctggtcagc tgcagctcgg tcctcctaga 83700 agataaaaac aaaccagcctcccagaagaa taacaaatat tcctgctgtg agccgcaggc 83760 acggaggagc tgtggtttccattttctttg gctctcccct cctgtgactt ctctttcagg 83820 ctgctgtaga ctccaggccctgctgacttc ctgctgctct gactcctctc catcttcccc 83880 cacctgtgag ctgtgctcctcccgatctca caatccctgt gttctctctt cacctgaaac 83940 tcactttctt cctttctctccatcttctct ggaagactca ggagacctcc ctgcagcctc 84000 aggcgtgtgt cgccaacctccctgcccctc tctgtccacc cagcacatgg gacacagaac 84060 agcctgtctg ggcttatctattttcatcaa gatgaatggg ctcctgcact agatgtgcaa 84120 agatgtaccc ctttcactcgcaaggctgca cctagttctc tttttatgga agactctgag 84180 atgtcctaac atactcttcctaaaatactt gtcctaataa ggtgcttcta acttccgaaa 84240 gaaagcccta aaaaccttccttggtatctg tgacgtgctg ggcatgtgct cagattgcat 84300 tgcccttcat cagttgtaactgcagtggcc atgttttcca aactgaaaat cagatgccta 84360 acaaattgcc ttcattgaaaataatcaaat tataagaaat tggtactgtc ccaggaaatc 84420 caggacacat gtccccagatgaaaagcacc tggttagggc taagccctct gcttctcctt 84480 tctctccaat ccaagctcaaacgtggttac cagagggcga gggagtgtac cctagttccc 84540 accacctcct ggttcccaatttgttgctgg aacaaaaaag tggccattag ccacacaggt 84600 ccctgagcag tcttggttctgtttccaaat aaaggccaag gtttccacct ccctaaggct 84660 cacatcagag gctcctctttctggaacctg gaagctccct gggcttattc tggaaaaatt 84720 gattgtccct gccacatagaatggaagaag actctcgggt tattctagga acctcagtga 84780 gtttatctca gaatttggatgatcattccc actgaatggg aacgtgattg aaggaggaca 84840 ttgtggttct tattaacacaatatcaatga ctgtcatcct gcctctctca tttcccttcc 84900 tcatctccta gagccctcatataaggcagc aggggcaagc cagccacagg tgcattcctc 84960 gctctttcct tgtcttcttgttccattcag gcccgaatct tcaaattgct cctgtcttct 85020 ccttctaaga ggtgccacctgcagtgggtc atgctcctgg ctgatgctgc tagaaaaaga 85080 actcatctgt atcactggcggtctttcttt tccttccagg ttttcatggt ttgtgtcccc 85140 aacaactcat ccctgtcctcccatcttgca gactgcctgg tgtgaccaca ccgacagact 85200 gattcctgca gccggctctggtcctcccta ccccgggggt cacagtgctt ccttcccttc 85260 gctccctttt aataagatcttaagactctt aaagtaaacg tattaagcaa gatcatcctc 85320 atttgatgta tagaaaatttaggcttacac acttcagctc aacattagaa agtttacaat 85380 taggccgggc atggtggctcacgcctgtaa tcccagcact ttgggaggcc aaggcaggcc 85440 gatcacgagg tcacgagtttgagaccgtcc tggccaacat ggtgaaaccc cgtctctact 85500 aaaaatacaa aaattagctggtcgtggggg catgtgccta tatcccatct acttgagagg 85560 ttgaggcagg agaattacttgaaccaggga gttgtaggtt gcagtgaacc gagatggtgc 85620 cactgcactc cagcctggcgacagaatgag actccgtcta aaaagaaaaa agaaaagaaa 85680 ctttaaaatt aaatctgtagaatggactgc cagcaaaaga agtgagtatc ccatcaacag 85740 aaaggaccaa aaagaggctggatctttgga ggcaggaact gcctaatcta aggtgtcatt 85800 gattgtacga cactattattttaaatactg gctgggcaca gtggcacaca cctataatcc 85860 cagcactttg ggaggccgaggtgggcagat cacttgaggt caggagtttg agatcagcct 85920 ggccaaatgg caaaacccagtctctactaa aactacaaaa aaattagccg agcatggtgg 85980 cgcacacctg taattccagctagttgggag gctgaggcag gagaaccact taaacccggg 86040 aggcagaggt cgcagtgagccaagatcatg ccactgcact ttagcctggg tgacagagca 86100 agattccatc tcaaaaacaaacaaacaaac aaacctctaa gaaacaacaa caaaatgcta 86160 tcacttaaac aaagtgttttcacttaacct ctcatatttt gattgtaaga cattgcctga 86220 tttcacagat gttaaagagtgaaaaaaatg tgcaccttag acttgagaca taatagtgga 86280 ctttcacagt cctttccagatctaaccttt tattacttcc ttgaggctat tcagtgacat 86340 gtcagaccct ccttttgcttgaccttgagc tgccctgttg gaaaaaaagt gaatcaagtt 86400 caacattgct tctaaattgccctgggatag cgatactagc tccatttcca gtaaatagca 86460 actctgttct ctgtccctcttgcctggaaa caggtagctc ttcctcataa gtcagggctt 86520 ggcaaacttt ctctatatagggtcagatag taaatagttt aggctttgca agctacatag 86580 cctctaccag actctgctcaattctgctgc aggaatcata aagcagccac agataatatg 86640 taaacaaata aatagggctgtgttccaata aaacatcatt cgtggacact gaaatttgaa 86700 tttcacatca ttctcacttcatgaaatatt attcttcttt tgactttttt ccaaccatgc 86760 aaaaatgtaa aacacaaaacaatacttagc ttgagggcag catgaaagta ggcagtgaac 86820 tggatttagt ccttggactgtaatttgctg acctcaatct taggcagtcc gtggctacaa 86880 tagcaagttc tgttaatttcagtatgcctc agcctcctca acggtaatat ggagattgta 86940 atagcaccct ctcttgaagatttgtcatgt taattagatg aattaatgca tataaatcac 87000 ttaggacagt tcctggcacacagtaggcac caatatgtga tagctattgc tgctgctgtt 87060 gctgctgctg ctattaatttcacagtgctc cccccgcctc catacctagt gtccaccctt 87120 tggactgggc agcccagctttccctgtaat gaggaagcat gtgctgccct cttctggcag 87180 gcagaggaat cgtcgccagagccagctgat gtgatttatt caaggcctgt ggattcgaaa 87240 gggtcttcct atacaaagattcagttttat ctaagccaaa gcttagacgg ttcaaacaag 87300 gctaggtctt cttcaaggctcaaagccacc ttccattatc tgtcccccac accaagggaa 87360 gggcatgtcc accaggcctgacggatctac cataggaggt ccctggctgc ccctcccaat 87420 gacaactacc cccaaatgcttctttctccc tttcctcttc tccttctaca acctcgagtc 87480 tttacattta cgaacttccttgacctaaaa taaaaaagtg cttgaaagga ttcagagcgg 87540 cctgggaggg ctgttagcctagaactaaca agccccttat tatcaaaggc cctgtgcttt 87600 cccccaggcg tcatgaaagccactgctgct ggggggtagg tggggaggag ggtgagggac 87660 agtgaagtgg ggcattttttccagattccc aggatgtggt gagccacgtt taaaaaagag 87720 tgtgtatatt tattattccaagccacttca atcataaggc gcttcatgcc ttctcccctt 87780 cctccctccc agcccccagcccccgtgtca cagtggccct gtctggaggg cctggccatc 87840 caggggatgg atgcggcaccccccttctca ttctgccttg tgctctgcca cgggcctgat 87900 gagaaagcta aggccgtgtgtaaaaagcag caggaatgtg aaacctaatt accctgtagc 87960 atgctcagaa acagagtcacttaggtggcc gtggggctcc atgtctactc tgttggaagc 88020 agttcttttg gaggacagttgggtcccagg aagaccgctg cagaggcctg ggagccccac 88080 ggtgggctgt tatcaggccatcttggtgaa ggctcctggg tggtcaggcc gacctccaca 88140 gcaatgcgga taacatgtgaccgtctcacc acttcctcat aaagcagcca ttgctgtctc 88200 ctttcaaaaa cgcctctggtcacaaaggga cccttcaggc tgccttcctt ggcaacccct 88260 gtgtgcaaat agccaactaggggtattttg tttgttggtt tgttgggtta aagttttgct 88320 ttgactagca ttcttgtaaggaaaaacaaa taacctaggc tcggtgggac ccacctcgga 88380 ggtctggtgg gctctgcacttgcatgtcct cgtctcctcc ctcatggagg gtccttgggg 88440 ctgagggcag gagggagaggagtgggcaaa tgcttcctct ggtccctccc atgagctgcc 88500 tgagggattc ctccagagggtctttgattt ggttcattat tgaggtggat gcttccaatt 88560 tataatttaa caaatcttgctcctgaaacg aatccttggg cagttgtaag gaaggcggtt 88620 ggtggtggcg tggcctgttgaatggggcag gcatggggag aagggggtgg ggagagctgg 88680 gccctgcgtg ggtgggcgcattcattagtg gaattttgct gtgtgggctc ctggactgga 88740 gtagagcgga gaccaccaacaccattccat gtggtggaat ttcccgctgt ctgaccagaa 88800 tgagttctct tggcctggcctggatgcaga tcaagacctg gggaataaag gaacgttaaa 88860 taactgattt gcacatgagttaaatgttct ttcacctctt cctaccgtta cctcatccta 88920 tgcaaccaac acctcttgtctttgtgcagt ctacacatag gaaccaaata cgtttctcaa 88980 tctctctctc tctccacacacacacacaca cacacacaca cacacacact tatgcacttc 89040 gtgtgtgttc tgatgttcttatgtactggc aaaggccatc acatacctcc ttagacaggc 89100 tcatttcaat taatctaaaagaaagtgctt acatgcaaaa ttatattcat gatgggacac 89160 aatatattca tgatgggacacaatatccca ctaagcacag gatatgcaga ccacagaagt 89220 acaatagtgt gtccatgggtgtgtgtgtct acatttcatt gtgtgtgtgt aggttgaaag 89280 tatcacagtt ggtttatacaattactacat tcataacccc ttgtctgtgg ttaggacgta 89340 gctttgagga aactttttccaagtttctgc tgacctttag gggagaatat ctctggataa 89400 gttaacctgt agaacagattccctcatttt tggtagagct ggggctgaaa atgacaagat 89460 gtgnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 89520 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 89580 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 89640 taatttgttg ggaaaagttgctacatggta actttggctt taaaacccca ggctttcttt 89700 caaatcctgc tccagcgccgtcttgcagga aggcactcta ccccatctca agcttgtttc 89760 caggctgagc cggagtagcctcatgagtta agacaggcac agacttaagg cttaagcata 89820 aagcagcact tcttttcattcctcttgtta gccagaccat tttctacctt gcgaatacat 89880 ttgaatcccg tgggggttttagaggaagaa taacccccat aggttcttag ttggaaggga 89940 cccctgtaac aaaagacagattaacaagag aaaaacaagc agaagtttat taacatgtat 90000 attttctata atgaggagttctcaaagaga tggctttgaa ttctagtttc acagcatcct 90060 catcaaagga cagtaaacttttagagatgt gacaagggaa aaaacacttt gagtctctag 90120 gggcagcaac ttcgggggatagcaaatgaa tggcagataa aggccagtta gcaaagcttg 90180 ttcaatgtag attcctgcagggccatctcc aggagcataa gcgtcttaag ttgttttcag 90240 tggttctccc tggtagaaggggggaggcaa gatacgtttt gtctttgtaa atctacgtcc 90300 tgcttttaga caaatagagggagggcagag agctttactg cctctgtttc ttctgaattg 90360 tcttcatctc aacaatccttcctattttgg gggtggcata tcctggtttc ccacagggtc 90420 ttgttacgaa gcagatattgaggtagtgga tctggggcaa ggcctgagag ctcccaggcg 90480 attcggacac tgttggtccagggctgtgct gtgagaaaca gggttctgag cagctgtctc 90540 tgtgaggaag catgctgggtgtgcctcctt catcattgtc accccagacc ccagctcagc 90600 atctgaccca gggcagatggtccatcaata tttgttaaat gagagaaaaa aaagtataca 90660 aacacttcaa cacacctctaccttgtctat gcctttgctt gggagatgat gacggggcct 90720 catcagagcc acgggcacgtcctccagcca cttcctgtcc ccgctgtgga acctcacctt 90780 ttaaatctgt ccccatgttgcagactcccc tatgctctga ctgtatttat ttaaaacttc 90840 ccagtcccca tttaaatcctatgatgttct gtgactagca ttgtctttga cgtgagaaca 90900 tacaatgctt ttgtagcctctgttatatga tatcaagctt catggtgaat aatctcactt 90960 agaaccaatc aataaaagaatgcaagaacc tgagcagctc tcctcactag gatggccagt 91020 gtgctggcgg gcttcgctgaagtgggattt ttcttttcac cattattttt ataaccacat 91080 tcaagggaaa ttctatttggtgttaaaagt gatacttcac caagagtcaa aagagaaaac 91140 acagaaggct aaattctagacttttttcag ctagcagcca gaggtcaaat tatttttcta 91200 agacccctgg tggacaaagcatttaatgat ctggatcaag gtaaataaaa tacgtttctc 91260 tggccgggcg cggtggctcatgcctataat cccagcactt tgggaagccg aggggggcgg 91320 atcacaaggt caggagttcaagaccagcct ggccaatatg gtgaaacccc atctctacta 91380 aaaatacaaa aaaaaaaaaaatttagccgg gtgtggtggc gggtgcctgt agtcccagct 91440 actcgggagg ctgaggcaggagaatgccat gaacccagga ggtggagctt gcagtgagcc 91500 gagatcacac cactgcactccagcctgggt gacagagcga gactccattt caaaaaaaat 91560 aaaaaaaata aaatatatatatatatatgt atatgtatgt atatatgtgt gtgtgtgtgt 91620 gtatgtgtgt nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91680 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91740 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91800 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91860 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91920 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 91980 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92100 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92340 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92400 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92460 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92520 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92580 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92640 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92700 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92760 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92820 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92880 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 92940 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 93000 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 93060 nnnnnnnnnn nnnnnnnnnnnnnttgctac cctgaaaata tatatatata tgggctctgt 93120 cttctactag taacaaataaaaggtagact gaattcttgt atattcaatt cttatatatt 93180 cactttcaag ttaaggtgatattttttctc tctctggtga aaatacctgt cctagccatt 93240 gccaaaaagg acacactattttaaattctc ctttggtgct ggaaaaatct gagtcattta 93300 ccccctacta gatttcaggaaacaggaagt ataaaattgc acatttaatt tgctacacac 93360 ttcaaacttt aggagatgcaagactcatgt agtaaaagct gtgcaactca aacatccaat 93420 gggtagcttc tccccacaaactgtaatttt tgttggggca cattagggta tccagggaac 93480 tgctctgagc aagggggagggaggtccagg tgtacttgta gcatatctgg ggaattctgt 93540 gtaagtatca gtcacccccaacctttaagg ttgccccatt tcacatctgt tatgatgaag 93600 gctttcggct gggggactgtggttctggct gtagagtcag aatgaaagga acagctgaat 93660 atccctgttc atgtcagcatttcagggcag aggatgagtc aagcacgatg tttactgagc 93720 agagccagag gcaagagccacttttttctt gcagctctaa gaattttttg ttgctttttt 93780 tgtatgcttt gtttctcatacatcagcatc atcatgcgtt gcacatacac attgtacata 93840 acaactgctt acagcatgccgggcactgta ctaagccctc tgcaggtgat gactcaatta 93900 ttgctcccag caaccctctaaggtatgcca tgttatgatc tccactttgc agagaaggaa 93960 acaagcaccg gttaagcaacctgcccaagc tagtatgtgt cagggtcagg accccagccc 94020 aggcagctgg tcccatgatctgtgctcttg acttgcgtat attaacattt aatataatcc 94080 actatttaat atgtatgtagtgctgaatta aacaagagtg tgtcatttag gaaatggtga 94140 tcaaatcagt tcacgatccgatgaatactc aatgaatgac taccatgtgt atttccttaa 94200 atatactcaa cacttcccaccttccccatc agggtcctca cccagcaatc catgtgggga 94260 aaggggcaca aagttggcccacagtgggca ccgaatagat gcctgtttaa caaataaaag 94320 aatgggcaac tggaagagaatttgtaggat attcacagaa tcaccaaatc cagtaattat 94380 ttcagtctag cattgcagtggcacagaggc agtttttaag tttgtgaatg gctcttatgt 94440 aaaagatgtg gctatttgaggatgaaacaa gacaaaatgt ggtgaatgta cttcagccta 94500 agtgttttgg gggagggagaattgagaaaa atctctaggt ctaaacgctg gaacggatga 94560 ctgataaagt tagaatctctcttaaccagc tcattctaag tgtgtgagca tccgcattaa 94620 acctaatcta tggcctgatacttaaaaata acctgttata aagtaaccat aaaaaaactc 94680 acagatttca gcctgattcatttggccaca taaactacaa tcagcattaa tagcttccgt 94740 cagcatttat gccgtactttctattagcac gatgggttct acaatcattt atgagtttct 94800 ggaaagtcta atagaaaacacacctacatg ttctctgtgg aaaatacaca ataggttttt 94860 ggggagtttt ttgtttgtttgtttgtttgc tcagggataa gccagaaggt gtcgccactg 94920 ccccatggct aaagtctgggccgggttccc taaccagaac ttctcccctg atcactcctg 94980 agctcgctag ctctgcagcttcctcccaca ttcagtcagt ctctctctct ctctnnnnnn 95040 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnttttaa acatagccaa atactggcca 95100 aagtctcttg cctttaaaaaaaatatgcat tttgttgttg tactaatact tctgaacata 95160 cgccaaatat aagggtgatttgtggagctg tattaaataa aaagatgctt ctttataaga 95220 atcccctata gagcttggggaagcagctat gtggagaatc tgtgtcttcc ttaagaaagt 95280 ttctgaattc cttggaaatacatcagaaaa caccccattc tccccaccac agaggagggt 95340 gggcaaccaa cagtgtcctatatcccaacc caccataatt gtaagactta aacgctatct 95400 ttcaaataaa gaatcatttactggcttctt actggaaaga actataggag cttgtcattt 95460 aattcatcct cccctctgaggctgaaattc tttctagaac aaacaaataa tacccatggc 95520 accacgtgct gcacactctctgtagcccag ctgacgccac acgagggaac agtggggaat 95580 ctgttgaagt tgccattgcattttgctttc tcataaaccg ctgtttgtct ttctgccttt 95640 cctgttttta gcagtgcctagaaacggagg cctgggaatt gcctccccca tgtgagcccc 95700 tgggttgttc acacccaaggccaatttgac aacatcctcc tactctggct atgaggccaa 95760 gtccagtgtt ccttggttcagtgccaaagg tggacatgaa ctctctaaac tgtcaaacta 95820 agaaaatgtg acacacccatgtggcctgga gagagcaaag ccaaacccaa agagatgctt 95880 accaggcaac tttagatccctaacatgtta ggaaagtcaa gagcaagctt catcctgcaa 95940 gctctccagg attctccttggacctggaac atgctaggat gttcagataa acttcacttc 96000 agacactgaa tttctgccttgagctatctc ttctctcatc tttctctcgg tgtcttcttc 96060 cccttcatcc tctcataaacccaagttcta tagctgtaat aataataata gcagttaact 96120 ttaatcaagt ctcactctgcaccagacact agatgatgtg aacctgtgaa caagcataac 96180 tgaggattca aacccaaatctcttatggca gagtgaatgt gctgctctgc tactgtgttt 96240 gttatcagaa gccatggctccccgacatac tcgacccaga ttctctgttt cttccttgat 96300 gaaaccagtg acatgcaaacatagccagag ggcccaggca cagcagagag cgtcccatca 96360 gacctgatag aaagtccccttcccatcttc taaaaccttg ctgccatttc tgacttcatg 96420 tggtctaaga atctggtgtagacattattt aaaagacaaa gggacctgaa aatcagcccc 96480 cccttacctg ccatgagtcctagaaaggtc actgcggatg agaaaacccc agtttccacc 96540 tgctttataa ttttgcctttgtgtggtcat tcatactcat ataacctaac tgtaactggg 96600 ctactgttct ctttctttaaggactttggc aactgagaaa aaagctacat gactcatttc 96660 taaaacctga aagatgaatgcagatcttta tgtaaagttt tctggtgggg gaggggattg 96720 gttacactgc ccatcacagagctgaacatc ctcaaaatcc ccttagaata tctttgcctc 96780 tcctcaaaat tgagctcgttatagcagcat ctacatttct gcctgcttgg cctgcacctg 96840 cccttttcct caccctcccaatcctcctgg gccctgctgg ggctcctaca tcccgcttgc 96900 tcctggatct ttggcctagtccatctgatc tctctcagtg tctcttcctc ttgtcctagc 96960 tgcctgtgga aagaaaggcttcttcttcca acagatgagt aagtggtgat attcttctta 97020 ttattatttt ttgtttccatcacgttacca ttcacgtttt aaactatcaa atgtcaacaa 97080 gtgtggccat ctttgtattttctcttcaaa aatatgtgat ggccagagac atctccgtct 97140 gccatggtct ttctcaaaccaccactgtac tttttttgca ccctttggtt atatcaacat 97200 gatctcaaat gagaagatgaaaagtaaaaa cttcattcag tgctccaggt atttctcaat 97260 atctagatat ttctttcctcctaggaaaaa tccaaatgga tttctcactt tttgaccaga 97320 tgtttcattg tcacacagcccgtgtgacag cattggcccc tagtacaggc agagttgaaa 97380 agtgatgtga tgggtcatggttgcccctct actcagagaa aactccttct tgacctcatc 97440 acggtcgctg aaagtactaagaattatatc tgaaatcaag cactggtagc agaacacaaa 97500 tatgttacca aattagttggaaggtaaggc atgggggagt catgagggga gccccatcct 97560 cttttgtcag gggaatgagaacattttgca gcacctgtca ggccacgtga atacccaagg 97620 agtgatggga cagtccttgcacccagaagt ggccttgcag agtagaagga gtttatgttt 97680 tagaatctaa tagccctgcgcttaaatcct gggctttgca cctaaattat aatagagcta 97740 atcgattgaa catttttttctctctgtcac tctgagtacg tgattctaat ctgccctagg 97800 cattcattga cctcagagagattaggattt ctgtaaaagg aactctaaga cagaaaaaag 97860 aattcaatat tcagcttttgatcttcaaaa tagcattttc aacaagacag tagactttta 97920 cccaacctat tgtctaagatgttctaaact cttttatttc tcaattttta cccatcaaaa 97980 accctgcctg gaggcctcgactcacagagt agaaacagtt gtactggcta tgcagcaagg 98040 ccgcgaggct ttctgctcaatgattggtgg gctatttgac ctgggaaaac taaaagaaaa 98100 gcccaccctt cctagcacaggggagttggg gatgaagaag aaaggagagg tggtgggggg 98160 gagggtcagg gaactagagcatgttaagag ctccgtgtgg tgctttgctt gggtatcctt 98220 ccagattgtg gagggaggcaagcagcccac ctgtaacccc cacccagcat ccccatggtt 98280 actctggaag gaagcaaacagaagaaatgc agaacctgta ttcgctttcc aaagccttcc 98340 acccatagat tccagaaaccccatctcccc agtgctgtga ggtcacaggg gcctagtccc 98400 caattcccag tgccactgactcaggaagga gagcaaggag gggcaggttc tctgggaaac 98460 tgagcatatc aatgcaaagagacagaattc tctaggggga ctatttagat gattacatca 98520 gactaggtta taatccaaaatggacgaaat taaagttgtt gttgtttttc caccacccag 98580 aggttaggag cttgtaaggaagaccatagc agttgaaaac aaagcaaggc acatttccat 98640 tgtacgtccc aggagtagtttgaatggttt aaggaccagc tatgtgtgtg cttgcataag 98700 cgtatgtgcc ctgggtaagtctaagactca gtttcttcat ctgttgaatg acgatgaata 98760 atattttcca tctcaatagcctcattgtgt gtttaaatgg tagttgcata aacatcccta 98820 gctcactgcg ggaattcagtgagtgtattt ccttgctggt gctaaggtat gggactaggg 98880 caataaaagg aacaagatagggcagagggt actcggccca ggccagtgcc caataaccac 98940 aacctccttc tccatggagagttgaacaga cacccatcca atctacagac aacggtcaca 99000 ggtgggttcc tggtttttattttgctgcaa cacacataca agagaaaaac catatatgag 99060 aagttgagcg tgcagccacctgggcccaca gagctgttct cattctttca aagaccacct 99120 ttgatatctg ctcctctccatgtgaatttt gagccccagg agtctgcttt gccttatgct 99180 catcaggacc tgtgctgcactgagtttgcc agcaaaccag ggctgagctg ttcttgcaag 99240 aacttgttgg aggctgggttagagtcttgg ctccccagtc cccattcagc ctccatgccc 99300 tggtaagaat atattattttcttagctcaa attgctgttt ccaagtcatt atttctctac 99360 gacgagaaaa caatattgggtcttatgtaa tatcaacctc aatgaatctg gaagtgatca 99420 tggaatgggt ggattttttttcctccttaa aaaagagaaa aataaatcta agattgtttg 99480 catatggctc agaatatttatgaactattc cccttgtttc ctcatcacat attttaccca 99540 gtgcatttgt gtgtggtacagaataaatct gtatggggag cagaggcgga gaggggaaga 99600 aagcagttaa aacactgggtatctatcaag tccctgggat gttagtgatc ctgcgtcttg 99660 ggtgccccaa ccctaggctcacttgctgta gttggaggga tacccccatc tagctactca 99720 gagtaattat gtggagtgaggatttcaaat atttacgagg aattaaacac aaagaattaa 99780 catctttttg ttgctctagtttctttcaca tcccccttgt tcctaccaga gcagaaagtt 99840 caaatagttt catgacctgtgtaccatcag tcaaaagaga tcattaaatt ctatggatgg 99900 cagattgagc cagggtcagaggaatgagtt ggcaggtggg aggaaaagaa ttggcagagc 99960 gtatagtggg tactgccatttgctatccga taatatggta gtgtttcagc acacaggcat 100020 gttgtgtcta ttttgctatgggtgtgcctt tcaacacagc ttatcaccaa ggagcatccc 100080 aagggagtat aaggaacagggctttggctg tatactaaat aagtaaggga agcaaatctc 100140 caggctattg attttcctctccagggagtt ggccacaaat ttggagcttc ttaaaatcac 100200 ccattctcca ctgcaccttgggatattcag gaatatattg accaagagta tggggcaagg 100260 aacagagaac aggaggaccccacgtttggt aaggcaatag caacaactac cattaattga 100320 tgtcatcgta ttggccaggcactgtattag gtgctttccc tgcattatct gagttaaccc 100380 cagcagtggt cttaacagggtgaaggatcc ttagtcccgt tattgagatg agaaagcaga 100440 aactcagtga agtagaggaacttgtccaag gtcacatgta agtgccagga tcaacgacat 100500 ttgtgggatt tatgaagccttaaggcaata caataagtac caatacagaa ttcttgaaat 100560 cagatccaag cgcttggaagggacccagga aagcaagaca ccttgaagct taaggctcag 100620 cagcttccca gtgaattcaccctgggcggc cttgagattt aaacacagtt tagtcccagg 100680 ccgtgcagtc ctttccctgttgccttgtct cctcttcctc taccacggct gttaccaact 100740 gcagccctca cattcctggtcaaaaataca caaacccaag tgaggagact gatacacaga 100800 gggtgccttc cagaatgtgctgacttggaa agctccatgg aatgtcacag ttgcaacatg 100860 aatcaacaat gattcctgtcaccagacttg atgccttcca tctcgaggaa ttaccggagg 100920 gatataaaga actatcttcccaaggaactt caatcactgc tctaggcttt tcaccaaatt 100980 tcatgatatt tcccagacctggtgacatgg gcacagactt gtgcaacagc ccactcaggt 101040 ctccagggcc atttgagattagaacctgca atgtctcatc tcctatcgta tgactctttc 101100 cctcagatga tattagttggtcaaattttg acaggcccag tatttgattc ttcctttctt 101160 cattataatc aagattttgctgtaatctcc aggaatgaaa ggggtagagg gattgagcaa 101220 gcaaacaaat aaatcacaagtgcttcaggg agaaaagcat aataagcatt gtttagtacc 101280 caccctctgc cattgcctttgaatagcctt gcaatcccat cacagtctag acaattatta 101340 aggaatatac tgcattttcccagccctgct tactctgttc tcccaaactg gaaacacaaa 101400 gcagatgatg aagtagttcaaatgccacac cagggagaag aaagtattga gaaactatgc 101460 ccaacctcca ggcagttttcaaacattcca gtaagtgttc aattgattta tcttgggctg 101520 gtgtctttcc attgtctcaaacaaaaggcc ttctatgaaa agacaaaaag tgggttatga 101580 gaggagtggc taacaaggcacacttgggaa aatctgaaca agagtacagc cctcttggct 101640 ttgcctccag aaggcagtgtccgccttgta tctactggaa tgaacagcag gatcgacttc 101700 cattatctca gtgtcaggctctaaaccaga gtctggggat ggggctgatg acacaggtgc 101760 agtgactgat gccctggtcagccatctcct ccatgtttgt cctttgagct caagtagaaa 101820 gtttctatgg tagtgttctctgattagaaa ggactacgag attctgggca aaacgatttt 101880 cagtgatgat ggcttcccacagttgttctt ttgggtattt tgtctgcttt tgtgtctaaa 101940 taaattatgt gacagataattatttgacca agactgtaaa agccttctcc catgttatgt 102000 tcctttgggt gttcattccttgagtcataa tcatgacagc tttctgtgca gctgggggtt 102060 gcacatttgt ggtggagcccttccatttga gacagcagtg atttttagaa caagcttccc 102120 tagagtaacc caagtgttcttccctggtac ccatcacttc tctgggggtg acatcaaaaa 102180 gggctgttct attccccctgtcaagtcccc ctgaaggcct tgtccccaac tcttcttggg 102240 ttgggttagc tatagttaatacttccacaa tatctgatgc atatctttgt cttggaactt 102300 ctatatagtt tatatttacccatttaatag ccttttcttc ctaaaaatgt aatatgtagg 102360 ttgatgtgga atattagcaaattatagata accaaaactt tttaaaaaaa tggctagtat 102420 tgnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 102480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 102540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 102600 nnnnnnnnnn nnnnnattctcttgcctcag cctcccgagt agctgggtac tacaggcgcg 102660 tgccaccatg cccagctaatttttgtagtt ttagtagaga cggagtttca ccataatggc 102720 tagggtggtt tcgatctcttaacgtcatga tctgcccgcc tcggcctccc aaagtgctgg 102780 ggttacaggt gtgagccaccacacccagac tgtgtttcta tatttatgta tttttttcaa 102840 agcaaaagat tagctagtgtttattgtttg gttagagttt gccttttcag cctatttagt 102900 acacttctac tgcattgtgttaatggtttc caggtattct gcagtgtgtg gggagctggc 102960 agtgtgatga aggggacaggattctagatg agctactcca ctctgaagac cctatgactg 103020 tatccactgg caagaagtttgaataaaggc ccttttactc tttcatgcca gctcttgaaa 103080 gaggcctaac caaggctctaaagaaattgg atgactacct gaacacccct ctaccagagg 103140 agattgacgc caacacttgtggggaagaca aggggtcccg gcgcaagttc ctggatgggg 103200 atgagctgac cctggctgactgcaatctgt tgcccaagct ccatgtggtc aaggtaagag 103260 agctctaccc acaggggcctgcaagatcca gctccatctt aggcccaggt cacctgtgtg 103320 gatgagtcaa ggacagtaccacctgttggt caagaacctg gaccctgaag tcaggtaata 103380 aggacccaag gtcaccctctgctgcttgtt ggctgtgtgg cctccttgag cttcagtttt 103440 catttataaa ataggcatatattgcttact tcaagtgttg gtggaagagt aaatacagcg 103500 tgaaagtgct tggcatattgtgggggctta atatgtgtaa tagtcgcaat tatcgttgtt 103560 gtatacagtc atatcactccaaaggcctct tcctcatagg atttccctgg ctacacccct 103620 acagctctat taaatgtgccctcatatgca ttttttcttt gtgcacagac ccaccttctc 103680 acttcctcca gcaacttcctaaggtgagcc cacattattt tcctcatcta tcaaatgaag 103740 aggtggaggt tgcaagaagtgatgtcactt tcttgctatc attgcactta ctaaccattt 103800 gcagcatgta gtgtcatcctctcctatata acaaaccctg ggaatctgag agttggaaag 103860 gacatttaga ggtcatccaaaacaatctcc cacttcaacc tggaccactt tctgctgttt 103920 ctgtgacagg cttctgtgaagctgtgactg cagcctctga gaacgaggag ccctctgctt 103980 aatgagccag ccctaccatgttagatagct ctgattatta aatcattgtt ctttacaatg 104040 agcccaagca tgcctccctgctatccattt tttctctaga gtaacagaga acagctttgc 104100 ttgccttcac ctcatttgaagacagtagtt gtatccccct aagctctgtc aatgagcact 104160 tcttccccca ttctttcctgaccctcgtca gcctagtatc agatagccat actgtgctct 104220 attttacgca tgcttatatcttactgtctc agccagacag caaactctgt gaggaaagga 104280 actttttaaa gtgtgatggtgggcacacag tggccattca ataaatactc attgattgat 104340 catttgatca cctggtgtaagtctttacga tatccagttt atttctatgc ttcactgaga 104400 agactcagat tcaatcatctgtcagctgag tatatgccta ttatttttca gctcaagtcc 104460 cgagtcaaaa atgctatctcctctccaacc agagcatgta ccagcttgag ctgaagtcac 104520 tcagttgtgt gtacactggtgtttccgtat gccttagagc tgaagactga gagggaatca 104580 tgcataaaaa tggagtgggcaaatacaacc tatttagaaa agaacatttt ggatttgggg 104640 ccaagccaat agtcacttgtaggtccagcc aatctatgtc tctttgaagt tattaactac 104700 tgcatgcccc acccatcatcctttattctt cttctcctta ggaacaagta ccactgaaag 104760 ggatgatata attccagctcagtcacactg tgtcagagtg atacaatgca aagatcagga 104820 gacccgagtt ccggtcctgtatttgctgcc aactagcagc atgagctgag gcacatcatt 104880 taatcttttt ggaattcatttttctcgtgc ctagaagaac agaagtggat tgtattcctt 104940 cttgccttct tttcctttcttctttccctc cttctttcct tttctcttgc tcaaacatgt 105000 attcactacc actcaaaaaccatttgttga acaaagcaaa caaatgaatc tcccaagcct 105060 tgggcttcat cctgtgatttcctcaattcc cacctgcctt aaattactca gtgaagccct 105120 gtccttggag aaaattcagtgggtggttaa cccagagaag ctggagatca aaaagaagat 105180 ggccaatgaa agaacaaaggccagcccttg gcccctatct ctttggattt ctgctgatcc 105240 agcttatcag atcccagaaacctggcaaac ctctaaagtt cacaaagagc gaaggggaag 105300 ccaagtcagg cctccagtttggcttcggat gccaaaactt aatctgggct gtgggagcta 105360 actgttttca tatgaaagagcaaattcaga acatgagcat ggaagtccct gcgaacgtca 105420 gatctccgtg tgcatccttacccccttgct gctttcatgc tcactctcct cttgcgtggc 105480 tcgctttcag gtttatctccatccctggaa gcagagttgc tctggcccag gctctccatg 105540 agagtttggc ttgaacattcattgtctggc cccctcctag ttctcatctc ccaaagtcaa 105600 gccaatgtgt gaagaaatgaccagctcagc agccaaggcc cagggtgcac aggtcttcgt 105660 tgggagaggc atctgcaggcctttccttgc ccactgggat ccttgcctag catagtgacg 105720 atgttcagcc ctggagacaaacaagaaggg gaacaccaac atcaatagaa gtatatattt 105780 acaaattgca tttctgctgtattgaaacta acattctgcc ctttaaaatc ctgaaaataa 105840 aatttcagta tgaaatgacttgaggctact ctatgaatca gtgtgtcact gtgaaaaata 105900 cttttggatc cctttatcttattggagacc cttttcatcc actctgataa attccagcca 105960 gttctcttgg tcaggccaccactcctgcat gaatttgctc ttagccaaga cagcctcttc 106020 tcaaaggaac ttggcccaacccaagggatc atcatctttc agtgaacaga aagggactgg 106080 ggagatatcg tggtggcatctctcattgtg agagctttat caaaggactc ggacttcatc 106140 acccttgctt gtagttacctaagccagaca gaacagtgtg ggggtggctt ctttggtgcc 106200 cacaccaaac cagttattttaacagagaga atttaaggaa gtactatgta ctaaagaact 106260 ggaaaggcaa aatatactaggaggttctac cttcaagagg cagctactac tcctagggca 106320 gga 106323 4 197 PRTHuman 4 Met Thr Asp Ser Ala Thr Ala Asn Gly Asp Asp Ser Asp Pro Glu Ile1 5 10 15 Glu Leu Phe Val Lys Ala Gly Ile Asp Gly Glu Ser Ile Gly AsnCys 20 25 30 Pro Phe Ser Gln Arg Leu Phe Met Ile Leu Trp Leu Lys Gly ValVal 35 40 45 Phe Asn Val Thr Thr Val Asp Leu Lys Arg Lys Pro Ala Asp LeuHis 50 55 60 Asn Leu Ala Pro Gly Thr His Pro Pro Phe Leu Thr Phe Asn GlyAsp 65 70 75 80 Val Lys Thr Asp Val Asn Lys Ile Glu Glu Phe Leu Glu GluThr Leu 85 90 95 Thr Pro Glu Lys Tyr Pro Lys Leu Ala Ala Lys His Arg GluSer Asn 100 105 110 Thr Ala Gly Ile Asp Ile Phe Ser Lys Phe Ser Ala TyrIle Lys Asn 115 120 125 Thr Lys Gln Gln Asn Asn Ala Ala Leu Glu Arg GlyLeu Thr Lys Ala 130 135 140 Leu Lys Lys Leu Asp Asp Tyr Leu Asn Thr ProLeu Pro Glu Glu Ile 145 150 155 160 Asp Ala Asn Thr Cys Gly Glu Asp LysGly Ser Arg Arg Lys Phe Leu 165 170 175 Asp Gly Asp Glu Leu Thr Leu AlaAsp Cys Asn Leu Leu Pro Lys Leu 180 185 190 His Val Val Lys Ile 195 5197 PRT Rattus norvegicus 5 Met Thr Asp Ser Ala Thr Ala Asn Gly Asp AspArg Asp Pro Glu Ile 1 5 10 15 Glu Leu Phe Val Lys Ala Gly Ile Asp GlyGlu Ser Ile Gly Asn Cys 20 25 30 Pro Phe Ser Gln Arg Leu Phe Met Ile LeuTrp Leu Lys Gly Val Val 35 40 45 Phe Asn Val Thr Thr Val Asp Leu Lys ArgLys Pro Ala Asp Leu His 50 55 60 Asn Leu Ala Pro Gly Thr His Pro Pro PheLeu Thr Phe Asn Gly Asp 65 70 75 80 Val Lys Thr Asp Val Asn Lys Ile GluGlu Phe Leu Glu Glu Thr Leu 85 90 95 Thr Pro Glu Lys Tyr Pro Lys Leu AlaAla Arg His Arg Glu Ser Asn 100 105 110 Thr Ala Gly Ile Asp Ile Phe SerLys Phe Ser Ala Tyr Ile Lys Asn 115 120 125 Thr Lys Gln Gln Asn Asn AlaAla Leu Glu Arg Gly Leu Thr Lys Ala 130 135 140 Leu Arg Lys Leu Asp AspTyr Leu Asn Thr Pro Leu Pro Glu Glu Ile 145 150 155 160 Asp Thr Asn ThrHis Gly Asp Glu Lys Gly Ser Gln Arg Lys Phe Leu 165 170 175 Asp Gly AspGlu Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu 180 185 190 His ValVal Lys Ile 195

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 2. An isolated peptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 3. An isolated antibody that selectively bindsto a peptide of claim
 2. 4. An isolated nucleic acid molecule consistingof a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes an amino acid sequence shown in SEQ IDNO:2; (b) a nucleotide sequence that encodes of an allelic variant of anamino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotidesequence that encodes an ortholog of an amino acid sequence shown in SEQID NO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids; and (e) a nucleotidesequence that is the complement of a nucleotide sequence of (a)-(d). 5.An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids; and (e) a nucleotide sequence that is thecomplement of a nucleotide sequence of (a)-(d).
 6. A gene chipcomprising a nucleic acid molecule of claim
 5. 7. A transgenic non-humananimal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acidvector comprising a nucleic acid molecule of claim
 5. 9. A host cellcontaining the vector of claim
 8. 10. A method for producing any of thepeptides of claim 1 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 11. A method for producing anyof the peptides of claim 2 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 12. A method for detecting thepresence of any of the peptides of claim 2 in a sample, said methodcomprising contacting said sample with a detection agent thatspecifically allows detection of the presence of the peptide in thesample and then detecting the presence of the peptide.
 13. A method fordetecting the presence of a nucleic acid molecule of claim 5 in asample, said method comprising contacting the sample with anoligonucleotide that hybridizes to said nucleic acid molecule understringent conditions and determining whether the oligonucleotide bindsto said nucleic acid molecule in the sample.
 14. A method foridentifying a modulator of a peptide of claim 2, said method comprisingcontacting said peptide with an agent and determining if said agent hasmodulated the function or activity of said peptide.
 15. The method ofclaim 14, wherein said agent is administered to a host cell comprisingan expression vector that expresses said peptide.
 16. A method foridentifying an agent that binds to any of the peptides of claim 2, saidmethod comprising contacting the peptide with an agent and assaying thecontacted mixture to determine whether a complex is formed with theagent bound to the peptide.
 17. A pharmaceutical composition comprisingan agent identified by the method of claim 16 and a pharmaceuticallyacceptable carrier therefor.
 18. A method for treating a disease orcondition mediated by a human transporter protein, said methodcomprising administering to a patient a pharmaceutically effectiveamount of an agent identified by the method of claim
 16. 19. A methodfor identifying a modulator of the expression of a peptide of claim 2,said method comprising contacting a cell expressing said peptide with anagent, and determining if said agent has modulated the expression ofsaid peptide.
 20. An isolated human transporter peptide having an aminoacid sequence that shares at least 70% homology with an amino acidsequence shown in SEQ ID NO:2.
 21. A peptide according to claim 20 thatshares at least 90 percent homology with an amino acid sequence shown inSEQ ID NO:2.
 22. An isolated nucleic acid molecule encoding a humantransporter peptide, said nucleic acid molecule sharing at least 80percent homology with a nucleic acid molecule shown in SEQ ID NOS:1 or3.
 23. A nucleic acid molecule according to claim 22 that shares atleast 90 percent homology with a nucleic acid molecule shown in SEQ IDNOS:1 or 3.